Therapeutic RNA

Abstract

This disclosure relates to the field of therapeutic RNAs for treatment of solid tumor cancers.

Description

[0001] This application is a Continuation of International Application No. PCT/US2018/019878, filed on Feb. 27, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/464,981, filed Feb. 28, 2017; U.S. Provisional Application No. 62/597,527, filed Dec. 12, 2017; and European Patent Application No. 17306089.8, filed Aug. 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%.

[0005] Disclosed herein are compositions, uses, and methods that can overcome present shortcomings in treatment of solid tumors. Administration of therapeutic RNAs disclosed herein can reduce tumor size, extend survival time, and/or protect against metastasis and/or recurrence of the tumor.

SUMMARY

Embodiment 1

[0006] A composition comprising RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14 and RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27.

Embodiment 2

[0007] The composition of embodiment 1, further comprising RNA encoding an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24.

Embodiment 3

[0008] The composition of embodiment 1, further comprising RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9.

Embodiment 4

[0009] The composition of embodiment 1, further comprising RNA encoding an IFN.alpha.2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19.

Embodiment 5

[0010] A composition comprising: [0011] a. RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14; [0012] b. RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27; and [0013] c. RNA encoding an IFN.alpha.2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19.

Embodiment 6

[0014] The composition of embodiment 5, further comprising RNA encoding an IL-15sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24.

Embodiment 7

[0015] The composition of embodiment 5, further comprising RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9.

Embodiment 8

[0016] The composition of any one of embodiments 1-7, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment 9

[0017] The composition of any one of embodiments 1-7, wherein each RNA comprises a modified nucleobase in place of each uridine.

Embodiment 10

[0018] The composition of any one of embodiments 8-9, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

Embodiment 11

[0019] The composition of embodiment 10, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 12

[0020] The composition of any one of embodiments 1-11, wherein at least one RNA further comprises a 5' cap.

Embodiment 13

[0021] The composition of any one of embodiments 1-11, wherein each RNA further comprises a 5' cap.

Embodiment 14

[0022] The composition of any one of embodiments 12-13, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 15

[0023] The composition of any one of embodiments 1-14, wherein at least one RNA further comprises a 5' UTR.

Embodiment 16

[0024] The composition of any one of embodiments 1-14, wherein each RNA further comprises a 5' UTR.

Embodiment 17

[0025] The composition of any one of embodiments 15-16, wherein the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.

Embodiment 18

[0026] The composition of any one of embodiments 1-17, wherein at least one RNA further comprises a 3' UTR.

Embodiment 19

[0027] The composition of any one of embodiments 1-17, wherein each RNA further comprises a 3' UTR.

Embodiment 20

[0028] The composition of any one of embodiments 18-19, wherein the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.

Embodiment 21

[0029] The composition of any one of embodiments 1-20, wherein at least one RNA further comprises a poly-A tail.

Embodiment 22

[0030] The composition of any one of embodiments 1-20, wherein each RNA further comprises a poly-A tail.

Embodiment 23

[0031] The composition of any one of embodiments 21-22, wherein the poly-A tail comprises at least 100 nucleotides.

Embodiment 24

[0032] The composition of any one of embodiments 1-23, wherein at least one RNA comprises a 5' cap, 5' UTR, 3' UTR, and poly-A tail.

Embodiment 25

[0033] The composition of any one of embodiments 1-23, wherein each RNA comprises a 5' cap, 5' UTR, 3' UTR, and poly-A tail.

Embodiment 26

[0034] The composition of any one of embodiments 24-25, wherein [0035] a. the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0036] b. the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6; [0037] c. the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8; and [0038] d. the poly-A tail comprises at least 100 nucleotides.

Embodiment 27

[0039] A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering the composition of any one of embodiments 1-26 to the subject.

Embodiment 28

[0040] A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering to the subject: [0041] a. an RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 17 or 18; and [0042] b. an RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 29, thereby treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in the subject.

Embodiment 29

[0043] The method of embodiment 28, further comprising administering RNA encoding an IFN.alpha.2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 30

[0044] The method of embodiment 28, further comprising administering RNA encoding an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 31

[0045] The method of embodiment 28, further comprising administering RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 32

[0046] The method of embodiment 28, further comprising administering RNA encoding [0047] a. an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 26; and [0048] b. an IFN.alpha.2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 33

[0049] The method of embodiment 28, further comprising administering RNA encoding [0050] a. an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 12 or 13; and [0051] b. an IFN.alpha.2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 34

[0052] The method of any one of embodiments 27-33, wherein the cancer is a sarcoma, carcinoma, or lymphoma.

Embodiment 35

[0053] The method of any one of embodiments 27-33, wherein the cancer is a solid tumor.

Embodiment 36

[0054] The method of embodiment 35, 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.

Embodiment 37

[0055] The method of embodiment 35, 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, testicular tumor, carcinoma, adenocarcinoma, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.

Embodiment 38

[0056] The method of any one of embodiments 27-37, wherein the composition is administered intra-tumorally or peri-tumorally.

Embodiment 39

[0057] The method of embodiment 38, wherein the injected tumor and a non-injected tumor are both reduced in size after intra- or peri-tumoral injection into or near the first tumor.

Embodiment 40

[0058] The method of any one of embodiments 27-39, wherein the subject is human.

Embodiment 41

[0059] The method of any one of embodiments 27-40, wherein another therapy is also administered.

Embodiment 42

[0060] The method of embodiment 41, wherein the other therapy is surgery to excise, resect, or debulk the tumor.

Embodiment 43

[0061] The method of embodiment 41, wherein the other therapy is immunotherapy, radiotherapy or chemotherapy.

Embodiment 44

[0062] The method of any one of embodiments 27-43, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment 45

[0063] The method of any one of embodiments 27-43, wherein each RNA comprises a modified nucleobase in place of each uridine.

Embodiment 46

[0064] The method of any one of embodiments 44-45, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.), or 5-methyl-uridine (m.sup.5U).

Embodiment 47

[0065] The method of embodiment 46, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 48

[0066] The method of any one of embodiments 27-47, wherein at least one RNA further comprises a 5' cap.

Embodiment 49

[0067] The method of any one of embodiments 27-47, wherein each RNA further comprises a 5' cap.

Embodiment 50

[0068] The method of any one of embodiments 48-49, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 51

[0069] The method of any one of embodiments 27-50, wherein at least one RNA further comprises a 5' UTR.

Embodiment 52

[0070] The method of any one of embodiments 27-50, wherein each RNA further comprises a 5' UTR.

Embodiment 53

[0071] The method of any one of embodiments 51-52, wherein the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.

Embodiment 54

[0072] The method of any one of embodiments 27-54, wherein at least one RNA further comprises a 3' UTR.

Embodiment 55

[0073] The method of any one of embodiments 27-54, wherein each RNA further comprises a 3' UTR.

Embodiment 56

[0074] The method of any one of embodiments 54-55, wherein the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.

Embodiment 57

[0075] The method of any one of embodiments 27-56, wherein at least one RNA further comprises a poly-A tail.

Embodiment 58

[0076] The method of any one of embodiments 27-56, wherein each RNA further comprises a poly-A tail.

Embodiment 59

[0077] The method of any one of embodiments 57-58, wherein the poly-A tail comprises at least 100 nucleotides.

Embodiment 60

[0078] The method of any one of embodiments 27-59, wherein at least one RNA comprises a 5' cap, 5' UTR, 3' UTR, and poly-A tail.

Embodiment 61

[0079] The method of any one of embodiments 27-59, wherein each RNA comprises a 5' cap, 5' UTR, 3' UTR, and poly-A tail.

Embodiment 62

[0080] The method of any one of embodiments 60-61, wherein [0081] a. the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0082] b. the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6; [0083] c. the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8; and [0084] d. the poly-A tail comprises at least 100 nucleotides.

Embodiment 63

[0085] A codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 11.

Embodiment 64

[0086] The DNA of embodiment 63, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 11.

Embodiment 65

[0087] A codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 13.

Embodiment 66

[0088] The RNA of embodiment 65, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 13.

Embodiment 67

[0089] An RNA produced from the DNA of any one of embodiments 63 or 64.

Embodiment 68

[0090] A codon-optimized DNA comprising or consisting of: [0091] a. contiguous nucleotides having at least 78% identity to nucleotides 1-984 of SEQ ID NO: 16; [0092] b. contiguous nucleotides having at least 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16; and [0093] c. nucleotides encoding a linker between the nucleotides of a) and b).

Embodiment 69

[0094] The DNA of embodiment 68, wherein the linker comprises nucleotides 985-1026 of SEQ ID NO: 16.

Embodiment 70

[0095] The DNA of any one of embodiments 68 and 69, wherein part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 16; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 16.

Embodiment 71

[0096] A codon-optimized RNA comprising or consisting of: [0097] a. contiguous nucleotides having at least 78% identity to nucleotides 1-984 of SEQ ID NO: 18; [0098] b. contiguous nucleotides having at least 81% identity to nucleotides 1027-1623 of SEQ ID NO: 18; and [0099] c. nucleotides encoding a linker between the nucleotides of a) and b).

Embodiment 72

[0100] The RNA of embodiment 71, wherein the linker comprises nucleotides 985-1026 of SEQ ID NO: 18.

Embodiment 73

[0101] The RNA of any one of embodiments 71 and 72, wherein part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 18; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 18.

Embodiment 74

[0102] An RNA produced from the DNA of any one of embodiments 68-70.

Embodiment 75

[0103] A codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 21.

Embodiment 76

[0104] The DNA of embodiment 75, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 21.

Embodiment 77

[0105] A codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 23.

Embodiment 78

[0106] The RNA of embodiment 77, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 23.

Embodiment 79

[0107] An RNA produced from the DNA of any one of embodiments 75-76.

Embodiment 80

[0108] A DNA comprising or consisting of: [0109] a. contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1-321 of SEQ ID NO: 25; [0110] b. contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 382-729 of SEQ ID NO: 25; and [0111] c. nucleotides encoding a linker between the nucleotides of a) and b).

Embodiment 81

[0112] An RNA produced from the DNA of embodiment 80.

Embodiment 82

[0113] A RNA comprising or consisting of: [0114] a. contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1-321 of SEQ ID NO: 26; [0115] b. contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 382-729 of SEQ ID NO: 26; and [0116] c. nucleotides encoding a linker between the nucleotides of a) and b).

Embodiment 83

[0117] A DNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 28.

Embodiment 84

[0118] An RNA produced from the DNA of embodiment 83.

Embodiment 85

[0119] An RNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 29.

Embodiment 86

[0120] The RNA of any one of embodiments 67, 74, 79, 81, and 84 wherein the RNA is transcribed from the DNA in vitro.

Embodiment 87

[0121] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-85, wherein at least one uridine is replaced with a modified nucleobase.

Embodiment 88

[0122] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-85, wherein each uridine is replaced with a modified nucleobase.

Embodiment 89

[0123] The RNA of embodiment 87 or 88, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.), or 5-methyl-uridine (m.sup.5U).

Embodiment 90

[0124] The RNA of embodiment 89, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 91

[0125] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-90, further comprising a 5' cap.

Embodiment 92

[0126] The RNA of embodiment 91, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O) ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 93

[0127] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-92, further comprising a 5' UTR.

Embodiment 94

[0128] The RNA of embodiment 93, wherein the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.

Embodiment 95

[0129] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-94, further comprising a 3' UTR.

Embodiment 96

[0130] The RNA of embodiment 95, wherein the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.

Embodiment 97

[0131] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-96, further comprising a poly-A tail.

Embodiment 98

[0132] The RNA of embodiment 97, wherein the poly-A tail comprises at least 100 nucleotides.

Embodiment 99

[0133] The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-97, further comprising a 5' cap, 5' UTR, 3' UTR, and poly-A tail.

Embodiment 100

[0134] The RNA of embodiment 99, wherein [0135] a. the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0136] b. the 5' UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6; [0137] c. the 3' UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8 and; [0138] d. the poly-A tail comprises at least 100 nucleotides.

Embodiment 101

[0139] A DNA comprising or consisting of: [0140] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11; and [0141] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein when transcribed, the nucleotides of parts a) and b) form a single transcript.

Embodiment 102

[0142] A DNA comprising or consisting of: [0143] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1-984 of SEQ ID NO: 16; [0144] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16; and [0145] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein when transcribed, the nucleotides of part a), b) and c) form a single transcript.

Embodiment 103

[0146] A DNA comprising or consisting of: [0147] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21; and [0148] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein when transcribed, the nucleotides of part a) and b) form a single transcript.

Embodiment 104

[0149] A DNA comprising or consisting of: [0150] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28; and [0151] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein when transcribed, the nucleotides of part a) and b) form a single transcript.

Embodiment 105

[0152] A DNA comprising or consisting of: [0153] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25; [0154] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382-729 of SEQ ID NO: 25; and [0155] c. contiguous nucleotides having at least 80% identity to SEQ ID NOs: 1, 3, or 5, wherein when transcribed, the nucleotides of part a), b), and c) form a single transcript.

Embodiment 106

[0156] A DNA comprising or consisting of: [0157] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11; and [0158] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a) and b) form a single transcript.

Embodiment 107

[0159] A DNA comprising or consisting of: [0160] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1-984 of SEQ ID NO: 16; [0161] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16; and [0162] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b) and c) form a single transcript.

Embodiment 108

[0163] A DNA comprising or consisting of: [0164] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21; and [0165] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a) and b) form a single transcript.

Embodiment 109

[0166] A DNA comprising or consisting of: [0167] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28; and [0168] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a) and b) form a single transcript.

Embodiment 110

[0169] A DNA comprising or consisting of: [0170] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25; [0171] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382-729 of SEQ ID NO: 25; and [0172] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), and c) form a single transcript.

Embodiment 111

[0173] A DNA comprising or consisting of: [0174] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11; [0175] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and [0176] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), and c) form a single transcript.

Embodiment 112

[0177] A DNA comprising or consisting of: [0178] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1-984 of SEQ ID NO: 16; [0179] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16; [0180] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and [0181] d. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), c), and d) form a single transcript.

Embodiment 113

[0182] A DNA comprising or consisting of: [0183] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21; [0184] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein the nucleotides of part b) regulate the expression of the nucleotides of part a); and [0185] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b) and c) form a single transcript.

Embodiment 114

[0186] A DNA comprising or consisting of: [0187] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28; [0188] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and [0189] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), and c) form a single transcript.

Embodiment 115

[0190] A DNA comprising or consisting of: [0191] a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25; [0192] b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382-729 of SEQ ID NO: 25; [0193] c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and [0194] d. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), c), and d) form a single transcript.

Embodiment 116

[0195] An RNA produced from any one of the DNAs of embodiments 101-115.

Embodiment 117

[0196] The RNA of embodiment 116, wherein at least one uridine is replaced with a modified nucleobase.

Embodiment 118

[0197] The RNA of embodiment 116, wherein each uridine is replaced with a modified nucleobase.

Embodiment 119

[0198] The RNA of any one of embodiments 117-118, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.), or 5-methyl-uridine (m.sup.5U).

Embodiment 120

[0199] The RNA of embodiment 119, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 121

[0200] The RNA of any one of embodiments 116-120, wherein the RNA further comprises a 5' cap.

Embodiment 122

[0201] The RNA of embodiment 121, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 123

[0202] The RNA of any of embodiments 116-122, wherein the RNA is substantially free of double-stranded RNA.

Embodiment 124

[0203] The RNA of any of embodiments 116-122, wherein double stranded RNA has been removed from the RNA.

Embodiment 125

[0204] The RNA of any one of embodiments 123-124, wherein the RNA has been purified via HPLC or cellulose-based chromatography.

Embodiment 126

[0205] A pharmaceutical formulation comprising any one of the DNA or RNAs of embodiments 63-125 and a pharmaceutically acceptable excipient.

Embodiment 127

[0206] A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one or more of the RNAs or DNAs of embodiments 63-125, or the pharmaceutical formulation of embodiment 126.

Embodiment 128

[0207] A method of producing a polypeptide encoding IL-2, IL-12sc, IL-15 sushi, GM-CSF and IFN.alpha.2b in vivo comprising administering to a subject one or more of the DNAs or RNAs of any one of embodiment 63-125, the composition of any one of embodiments 1-26, or the pharmaceutical formulation of embodiment 126.

Embodiment 129

[0208] A composition comprising at least two RNAs, wherein the RNAs encode different proteins, and wherein the RNAs are selected from the RNAs of any one of embodiments 65-67, 71-74, 78-79, 81-82, 84-100, and 116-125.

Embodiment 130

[0209] The composition of embodiment 129, wherein the composition comprises two RNAs encoding GM-CSF and IL-12sc.

Embodiment 131

[0210] The composition of embodiment 129, wherein the composition comprises three RNAs encoding GM-CSF, IL-12sc, and IFN.alpha.2b.

Embodiment 132

[0211] The composition of embodiment 129, wherein the composition comprises three RNAs encoding GM-CSF, IL-2, and IFN.alpha.2b.

Embodiment 133

[0212] The composition of embodiment 129, wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-2 and IFN.alpha.2b.

Embodiment 134

[0213] The composition of embodiment 129, wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-15 sushi and IFN.alpha.2b.

Embodiment 135

[0214] The composition of any one of embodiments 130-134, wherein the RNA encoding GM-CSF is selected from the RNA of any one of embodiments 84-85.

Embodiment 136

[0215] The composition of any one of embodiments 130-134, wherein the RNA encoding IL-12sc is selected from the RNA of any one of embodiments 71-74.

Embodiment 137

[0216] The composition of any one of embodiments 131-134, wherein the RNA encoding IFN.alpha.2b is selected from the RNA of any one of embodiments 77-79.

Embodiment 138

[0217] The composition of embodiment 134, wherein the RNA encoding IL-15 sushi is selected from the RNA of any one of embodiments 81-82.

Embodiment 139

[0218] The composition of any one of embodiments 132-133, wherein the RNA encoding IL-2 is selected from the RNA of any one of embodiments 65-67.

Embodiment 140

[0219] A method of treating or preventing solid tumor cancer comprising administering a therapeutically effective amount of each of the following RNAs directly into a tumor: [0220] a. an RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); [0221] b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); and [0222] c. an RNA comprising an RNA encoding IFN.alpha.2b (SEQ ID NOs: 22 or 23), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 141

[0223] A method of treating or preventing solid tumor cancer comprising administering a therapeutically effective amount of each of the following RNAs directly into a tumor: [0224] a. an RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); [0225] b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); [0226] c. an RNA comprising an RNA encoding IFN.alpha.2b (SEQ ID NOs: 22 or 23); and [0227] d. an RNA comprising an RNA encoding IL-2 (SEQ ID NOs: 12 or 13), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 142

[0228] A method of treating or preventing solid tumor cancer comprising administering a therapeutically effective amount of each of the following RNAs directly into a tumor: [0229] a. an RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); [0230] b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); [0231] c. an RNA comprising an RNA encoding IFN.alpha.2b (SEQ ID NOs: 22 or 23); and [0232] d. an RNA comprising an RNA encoding IL-15 sushi (SEQ ID NO: 26), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 143

[0233] A composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acids of SEQ ID NO: 9, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 144

[0234] A composition comprising an RNA encoding an IL-12sc protein having at least 95% identity to the amino acids of SEQ ID NO: 14, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 145

[0235] A composition comprising an RNA encoding a GM-CSF protein having at least 95% identity to the amino acids of SEQ ID NO: 27, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 146

[0236] A composition comprising an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acids of SEQ ID NO: 19, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 147

[0237] A composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acids of SEQ ID NO: 24, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 148

[0238] An IL-12sc RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 149

[0239] A GM-CSF RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 150

[0240] An IFN.alpha.2b RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a uridine analog, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 151

[0241] An IL-15 sushi RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 152

[0242] An IL-2 RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 12 or 13, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 153

[0243] An IL-12sc RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 154

[0244] A GM-CSF RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 155

[0245] An IFN.alpha.2b RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 156

[0246] An IL-15 sushi RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 157

[0247] An IL-2 RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 12 or 13, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5' UTR (SEQ ID NOs: 2, 4, or 6), a 3' UTR (SEQ ID NO: 8), a 5' cap, and a poly-A tail.

Embodiment 158

[0248] The composition of any of embodiments 143-157, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.), or 5-methyl-uridine (m.sup.5U).

Embodiment 159

[0249] A composition comprising an RNA encoding IFN.alpha.2b, wherein the RNA is altered to have reduced immunogenicity as compared to un-altered RNA.

Embodiment 160

[0250] The composition of embodiment 159, wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.

Embodiment 161

[0251] The composition of embodiment 159, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.), or 5-methyl-uridine (m.sup.5U).

Embodiment 162

[0252] The composition of embodiment 161, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 163

[0253] The composition of any one of embodiments 159-162, wherein the alteration comprises a reduction in the amount of double-stranded RNA.

Embodiment 164

[0254] The composition of embodiment 163, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.

Embodiment 165

[0255] The composition of any one of embodiments 159-164, wherein the alteration reduces RNA recognition by an innate immune system as compared to un-altered RNA.

Embodiment 166

[0256] The composition of any one of embodiments 159-165, wherein the alteration comprises addition of a 5' cap to the RNA.

Embodiment 167

[0257] The composition of embodiment 166, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 168

[0258] The composition of any one of embodiments 159-167, further comprising a second RNA encoding a peptide or protein of interest.

Embodiment 169

[0259] The composition of embodiment 168, wherein the peptide or protein of interest is a peptide or protein selected or derived from cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, .beta.-catenin inhibitors, activators of the STING pathway, activators of the retinoic inducible gene (RIG)-I pathway, agonists of toll-like receptor (TLR) pathways, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis.

Embodiment 170

[0260] The composition of embodiment 168 or 169, wherein the second RNA is altered to have reduced immunogenicity as compared to un-altered RNA.

Embodiment 171

[0261] The composition of embodiment 170, wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.

Embodiment 172

[0262] The composition of embodiment 171, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

Embodiment 173

[0263] The composition of embodiment 172, wherein the modified nucleobase is N1-methyl-pseudouridine (m.sup.1.psi.).

Embodiment 174

[0264] The composition of any one of embodiments 168-173, wherein the alteration comprises a reduction in the amount of double-stranded RNA.

Embodiment 175

[0265] The composition of embodiment 174, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.

Embodiment 176

[0266] The composition of any one of embodiments 168-173, wherein the alteration comprises addition of a 5' cap to the RNA.

Embodiment 177

[0267] The composition of embodiment 176, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment 178

[0268] The composition of any one of embodiments 168-177, wherein the second RNA comprises: [0269] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0270] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0271] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; or [0272] d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 179

[0273] A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one of the compositions of embodiments 159-178.

Embodiment 180

[0274] The composition of any one of embodiments 159-178 for use in a method of treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis.

Embodiment 181

[0275] A composition comprising an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14.

Embodiment 182

[0276] A composition comprising an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27.

Embodiment 183

[0277] A composition comprising an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19.

Embodiment 184

[0278] A composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24.

Embodiment 185

[0279] A composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9.

Embodiment 186

[0280] A composition comprising an RNA encoding an IL-12sc protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18.

Embodiment 187

[0281] A composition comprising an RNA encoding a GM-CSF protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29.

Embodiment 188

[0282] A composition comprising an RNA encoding an IFN.alpha.2b protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 189

[0283] A composition comprising an RNA encoding an IL-15 sushi protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 190

[0284] A composition comprising an RNA encoding an IL-2 protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 191

[0285] A composition comprising any two of the following RNAs: [0286] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0287] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0288] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0289] d. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0290] e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 192

[0291] A composition comprising: [0292] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; and [0293] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29.

Embodiment 193

[0294] A composition comprising: [0295] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; and [0296] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 194

[0297] A composition comprising: [0298] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; and [0299] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 195

[0300] A composition comprising: [0301] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; and [0302] b. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 196

[0303] A composition comprising: [0304] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0305] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 197

[0306] A composition comprising: [0307] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0308] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 198

[0309] A composition comprising: [0310] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0311] b. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 199

[0312] A composition comprising: [0313] a. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0314] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 200

[0315] A composition comprising: [0316] a. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0317] b. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 201

[0318] A composition comprising: [0319] a. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0320] b. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 202

[0321] A composition comprising any three of the following: [0322] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0323] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0324] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0325] d. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0326] a. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 203

[0327] A composition comprising: [0328] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0329] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0330] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.

Embodiment 204

[0331] A composition comprising: [0332] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0333] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0334] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 205

[0335] A composition comprising: [0336] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0337] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and [0338] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 206

[0339] A composition comprising: [0340] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0341] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0342] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 207

[0343] A composition comprising: [0344] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0345] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0346] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 208

[0347] A composition comprising: [0348] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0349] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0350] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 209

[0351] A composition comprising: [0352] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0353] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0354] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 210

[0355] A composition comprising: [0356] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0357] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0358] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 211

[0359] A composition comprising: [0360] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0361] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0362] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 212

[0363] A composition comprising: [0364] a. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0365] b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0366] c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 213

[0367] A composition comprising any four of the following: [0368] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0369] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0370] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0371] d. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0372] e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 214

[0373] A composition comprising: [0374] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0375] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0376] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0377] d. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.

Embodiment 215

[0378] A composition comprising: [0379] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0380] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0381] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; and [0382] d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 216

[0383] A composition comprising: [0384] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0385] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0386] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0387] d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 217

[0388] A composition comprising: [0389] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0390] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0391] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0392] d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 218

[0393] A composition comprising: [0394] a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0395] b. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0396] c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0397] d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 219

[0398] A composition comprising: [0399] a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; [0400] b. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; [0401] c. an RNA encoding an IFN.alpha.2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23; [0402] d. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and [0403] e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.

Embodiment 220

[0404] A pharmaceutical formulation comprising any one of the compositions of embodiments 181-219.

Embodiment 221

[0405] A pharmaceutical formulation comprising any one of the compositions of embodiments 181-219 and a pharmaceutically acceptable excipient.

Embodiment 222

[0406] The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221, for use in a method of treating or preventing cancer.

Embodiment 223

[0407] The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of reducing the size of a tumor.

Embodiment 224

[0408] The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing the reoccurrence of cancer in remission.

Embodiment 225

[0409] The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing cancer metastasis.

Embodiment 226

[0410] A method for treating or preventing cancer comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.

Embodiment 227

[0411] A method for reducing the size of a tumor comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.

Embodiment 228

[0412] A method for preventing the reoccurrence of cancer in remission comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.

Embodiment 229

[0413] A method for preventing cancer metastasis comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.

[0414] Further embodiments of the present invention are as follows:

Embodiment A 1

[0415] A medical preparation comprising RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.

Embodiment A 2

[0416] The medical preparation of embodiment A 1, further comprising RNA encoding an IL-15 sushi protein.

Embodiment A 3

[0417] The medical preparation of embodiment A 1 or 2, further comprising RNA encoding an IL-2 protein.

Embodiment A 4

[0418] The medical preparation of any one of embodiments A 1 to 3, further comprising RNA encoding an IFN.alpha. protein.

Embodiment A 5

[0419] The medical preparation of embodiment A 4, wherein the IFN.alpha. protein is an IFN.alpha.2b protein.

Embodiment A 6

[0420] The medical preparation of embodiment A 2, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.

Embodiment A 7

[0421] The medical preparation of embodiment A 3, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.

Embodiment A 8

[0422] The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN.alpha. protein.

Embodiment A 9

[0423] The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFN.alpha. protein.

Embodiment A 10

[0424] The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-2 protein, and RNA encoding an IFN.alpha. protein.

Embodiment A 11

[0425] The medical preparation of any one of embodiments A 1-10, 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.

Embodiment A 12

[0426] The medical preparation of any one of embodiments A 1-11, 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.

Embodiment A 13

[0427] The medical preparation of any one of embodiments A 2-12, 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.

Embodiment A 14

[0428] The medical preparation of any one of embodiments A 3-13, wherein (i) the RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, 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: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, 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: 9.

Embodiment A 15

[0429] The medical preparation of any one of embodiments 4-14, wherein (i) the RNA encoding an IFN.alpha. 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 IFN.alpha. 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.

Embodiment A 16

[0430] The medical preparation of any one of embodiments A 1-15, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment A 17

[0431] The medical preparation of any one of embodiments A 1-16, wherein each RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment A 18

[0432] The medical preparation of any one of embodiments A 1-17, wherein each RNA comprises a modified nucleobase in place of each uridine.

Embodiment A 19

[0433] The medical preparation of any one of embodiments A 16-18, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.) or 5-methyl-uridine (m.sub.5U).

Embodiment A 20

[0434] The medical preparation of embodiment A 19, wherein the modified nucleobase is N1-methyl-pseudouridine (m1.psi.).

Embodiment A 21

[0435] The medical preparation of any one of embodiments A 1-20, wherein at least one RNA comprises a 5' cap.

Embodiment A 22

[0436] The medical preparation of any one of embodiments A 1-21, wherein each RNA comprises a 5' cap.

Embodiment A 23

[0437] The medical preparation of embodiment A 21 or 22, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment A 24

[0438] The medical preparation of any one of embodiments A 1-23, wherein at least one RNA comprises a 5' UTR.

Embodiment A 25

[0439] The medical preparation of any one of embodiments A 1-24, wherein each RNA comprises a 5' UTR.

Embodiment A 26

[0440] The medical preparation of embodiment A 24 or 25, wherein the 5' UTR comprises 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.

Embodiment A 27

[0441] The medical preparation of any one of embodiments A 1-26, wherein at least one RNA comprises a 3' UTR.

Embodiment A 28

[0442] The medical preparation of any one of embodiments A 1-27, wherein each RNA comprises a 3' UTR.

Embodiment A 29

[0443] The medical preparation of embodiment A 27 or 28, wherein the 3' UTR comprises 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.

Embodiment A 30

[0444] The medical preparation of any one of embodiments A 1-29, wherein at least one RNA comprises a poly-A tail.

Embodiment A 31

[0445] The medical preparation of any one of embodiments A 1-30, wherein each RNA comprises a poly-A tail.

Embodiment A 32

[0446] The medical preparation of embodiment A 30 or 31, wherein the poly-A tail comprises at least 100 nucleotides.

Embodiment A 33

[0447] The medical preparation of any one of embodiments A 1-32, wherein at least one RNA comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

Embodiment A 34

[0448] The medical preparation of any one of embodiments A 1-33, wherein each RNA comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

Embodiment A 35

[0449] The medical preparation of embodiment A 33 or 34, wherein [0450] a. the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0451] b. the 5' UTR comprises 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; [0452] c. the 3' UTR comprises 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; and [0453] d. the poly-A tail comprises at least 100 nucleotides.

Embodiment A 36

[0454] The medical preparation of any one of embodiments A 1 to 35, wherein the RNA is mRNA.

Embodiment A 37

[0455] The medical preparation of any one of embodiments A 1 to 36, which comprises a further therapeutic agent.

Embodiment A 38

[0456] The medical preparation of embodiment A 37, wherein the further therapeutic agent is an anti-cancer therapeutic agent.

Embodiment A 39

[0457] The medical preparation of embodiment A 37 or 38, wherein the further therapeutic agent is a checkpoint modulator.

Embodiment A 40

[0458] The medical preparation of embodiment A 39, 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.

Embodiment A 41

[0459] The medical preparation of any one of embodiments A 1 to 40, which is a kit comprising at least two containers, each container comprising at least one of said RNAs.

Embodiment A 42

[0460] The medical preparation of embodiment A 41, which comprises each RNA in a separate container.

Embodiment A 43

[0461] The medical preparation of embodiment A 41 or 42, wherein the further therapeutic agent is in a container not comprising the RNA.

Embodiment A 44

[0462] The medical preparation of any one of embodiments A 41-43, further comprising instructions for use of the medical preparation for treating or preventing cancer.

Embodiment A 45

[0463] The medical preparation of any one of embodiments A 1 to 40, which is a pharmaceutical composition comprising the RNAs.

Embodiment A 46

[0464] The medical preparation of embodiment A 45, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Embodiment A 47

[0465] The medical preparation of any one of embodiments A 1 to 46, wherein the RNA is present in a form selected from a liquid form, a solid form, or a combination thereof.

Embodiment A 48

[0466] The medical preparation of embodiment A 47, wherein the solid form is a frozen form or a dehydrated form.

Embodiment A 49

[0467] The medical preparation of embodiment A 48, wherein the dehydrated form is a freeze-dried or spray-dried form.

Embodiment A 50

[0468] The medical preparation of any one of embodiments A 1 to 49 for pharmaceutical use.

Embodiment A 51

[0469] The medical preparation of embodiment A 50, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.

Embodiment A 52

[0470] The medical preparation of any one of embodiments A 1 to 51 for use in a method for treating or preventing cancer.

Embodiment A 53

[0471] The medical preparation of any one of embodiments A 38-52, wherein the cancer is a sarcoma, carcinoma, or lymphoma.

Embodiment A 54

[0472] The medical preparation of any one of embodiments A 38-53, wherein the cancer is a solid tumor.

Embodiment A 55

[0473] The medical preparation of embodiment A 54, 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.

Embodiment A 56

[0474] The medical preparation of embodiment A 54 or 55, 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.

Embodiment A 57

[0475] The medical preparation of any one of embodiments A 1-56, wherein the RNA is for intra-tumoral or peri-tumoral administration.

Embodiment A 58

[0476] The medical preparation of any one of embodiments A 37-57, wherein the further therapeutic agent is for systemic administration.

Embodiment A 59

[0477] The medical preparation of any one of embodiments A 1-58, which is for administration to a human.

Embodiment A 60

[0478] The medical preparation of any one of embodiments A 44 and 47-59, wherein treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.

[0479] Further embodiments of the present invention are as follows:

Embodiment B 1

[0480] RNA for use in a method for treating or preventing cancer in a subject, wherein the method comprises administering RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.

Embodiment B 2

[0481] The RNA of Embodiment B 1, wherein the method further comprises administering RNA encoding an IL-15 sushi protein.

Embodiment B 3

[0482] The RNA of Embodiment B 1 or 2, wherein the method further comprises administering RNA encoding an IL-2 protein.

Embodiment B 4

[0483] The RNA of any one of embodiments B 1 to 3, wherein the method further comprises administering RNA encoding an IFN.alpha. protein.

Embodiment B 5

[0484] The RNA of Embodiment B 4, wherein the IFN.alpha. protein is an IFN.alpha.2b protein.

Embodiment B 6

[0485] The RNA of Embodiment B 2, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.

Embodiment B 7

[0486] The RNA of Embodiment B 3, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.

Embodiment B 8

[0487] The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN.alpha. protein.

Embodiment B 9

[0488] The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFN.alpha. protein.

Embodiment B 10

[0489] The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-2 protein, and RNA encoding an IFN.alpha. protein.

Embodiment B 11

[0490] The RNA of any one of embodiments B 1-10, 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.

Embodiment B 12

[0491] The RNA of any one of embodiments B 1-11, 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.

Embodiment B 13

[0492] The RNA of any one of embodiments B 2-12, 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.

Embodiment B 14

[0493] The RNA of any one of embodiments B 3-13, wherein (i) the RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, 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: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, 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: 9.

Embodiment B 15

[0494] The RNA of any one of embodiments B 4-14, wherein (i) the RNA encoding an IFN.alpha. 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 IFN.alpha. 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.

Embodiment B 16

[0495] The RNA of any one of embodiments B 1-15, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment B 17

[0496] The RNA of any one of embodiments B 1-16, wherein each RNA comprises a modified nucleobase in place of at least one uridine.

Embodiment B 18

[0497] The RNA of any one of embodiments B 1-17, wherein each RNA comprises a modified nucleobase in place of each uridine.

Embodiment B 19

[0498] The RNA of any one of embodiments B 16-18, wherein the modified nucleobase is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.) or 5-methyl-uridine (m.sub.5U).

Embodiment B 20

[0499] The RNA of Embodiment B 19, wherein the modified nucleobase is N1-methyl-pseudouridine (m1.psi.).

Embodiment B 21

[0500] The RNA of any one of embodiments B 1-20, wherein at least one RNA comprises a 5' cap.

Embodiment B 22

[0501] The RNA of any one of embodiments B 1-21, wherein each RNA comprises a 5' cap.

Embodiment B 23

[0502] The RNA of Embodiment B 21 or 22, wherein the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G.

Embodiment B 24

[0503] The RNA of any one of embodiments B 1-23, wherein at least one RNA comprises a 5' UTR.

Embodiment B 25

[0504] The RNA of any one of embodiments B 1-24, wherein each RNA comprises a 5' UTR.

Embodiment B 26

[0505] The RNA of Embodiment B 24 or 25, wherein the 5' UTR comprises 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.

Embodiment B 27

[0506] The RNA of any one of embodiments B 1-26, wherein at least one RNA comprises a 3' UTR.

Embodiment B 28

[0507] The RNA of any one of embodiments B 1-27, wherein each RNA comprises a 3' UTR.

Embodiment B 29

[0508] The RNA of embodiment B 27 or 28, wherein the 3' UTR comprises 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.

Embodiment B 30

[0509] The RNA of any one of embodiments B 1-29, wherein at least one RNA comprises a poly-A tail.

Embodiment B 31

[0510] The RNA of any one of embodiments B 1-30, wherein each RNA comprises a poly-A tail.

Embodiment B 32

[0511] The RNA of embodiment B 30 or 31, wherein the poly-A tail comprises at least 100 nucleotides.

Embodiment B 33

[0512] The RNA of any one of embodiments B 1-32, wherein at least one RNA comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

Embodiment B 34

[0513] The RNA of any one of embodiments B 1-33, wherein each RNA comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

Embodiment B 35

[0514] The RNA of embodiment B 33 or 34, wherein [0515] a. the 5' cap is m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0516] b. the 5' UTR comprises 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; [0517] c. the 3' UTR comprises 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; and [0518] d. the poly-A tail comprises at least 100 nucleotides.

Embodiment B 36

[0519] The RNA of any one of embodiments B 1 to 35, wherein the RNA is mRNA.

Embodiment B 37

[0520] The RNA of any one of embodiments B 1 to 36, wherein the method further comprises administering a further therapy.

Embodiment B 38

[0521] The RNA of embodiment B 37, 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.

Embodiment B 39

[0522] The RNA of embodiment B 37 or 38, wherein the further therapy comprises administering a further therapeutic agent.

Embodiment B 40

[0523] The RNA of embodiment B 39, wherein the further therapeutic agent is an anti-cancer therapeutic agent.

Embodiment B 41

[0524] The RNA of embodiment B 39 or 40, wherein the further therapeutic agent is a checkpoint modulator.

Embodiment B 42

[0525] The RNA of embodiment B 41, 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.

Embodiment B 43

[0526] The RNA of any one of embodiments B 1-42, wherein the cancer is a sarcoma, carcinoma, or lymphoma.

Embodiment B 44

[0527] The RNA of any one of embodiments B 1-43, wherein the cancer is a solid tumor.

Embodiment B 45

[0528] The RNA of embodiment B 44, 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.

Embodiment B 46

[0529] The RNA of embodiment B 44 or 45, 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.

Embodiment B 47

[0530] The RNA of any one of embodiments B 1-46, wherein the RNA is administered intra-tumorally or peri-tumorally.

Embodiment B 48

[0531] The RNA of any one of embodiments B 39 to 47, wherein the further therapeutic agent is administered systemically.

Embodiment B 49

[0532] The RNA of any one of embodiments B 1-48, wherein the subject is a human.

Embodiment B 50

[0533] The RNA of any one of embodiments B 1-49, wherein the RNAs are administered at the same time.

Embodiment B 51

[0534] The RNA of any one of embodiments B 1-50, wherein the RNAs are administered by administering a composition comprising a combination of the RNAs.

Embodiment B 52

[0535] The RNA of any one of embodiments B 1-49, wherein at least two of the RNAs are administered at different times.

Embodiment B 53

[0536] The RNA of any one of embodiments B 1-49 and 52, wherein the RNAs are administered by administering at least two compositions, each composition comprising at least one of said RNAs.

Embodiment B 54

[0537] The RNA of any one of embodiments B 1 to 53, wherein treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.

Embodiment B 55

[0538] The RNA of any one of embodiments B 1 to 54, which is or comprises one or more of the RNAs administered in said method.

Embodiment B 56

[0539] The RNA of embodiment B 55, which is or comprises one or more selected from the group consisting of the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN.alpha. protein.

Embodiment B 57

[0540] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein.

Embodiment B 58

[0541] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding a GM-CSF protein.

Embodiment B 59

[0542] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-15 sushi protein.

Embodiment B 60

[0543] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-2 protein.

Embodiment B 61

[0544] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IFN.alpha. protein.

Embodiment B 62

[0545] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein and the RNA encoding a GM-CSF protein.

Embodiment B 63

[0546] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-15 sushi protein.

Embodiment B 64

[0547] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-2 protein.

Embodiment B 65

[0548] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IFN.alpha. protein.

Embodiment B 66

[0549] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, and the RNA encoding an IFN.alpha. protein.

Embodiment B 67

[0550] The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN.alpha. protein.

FIGURE LEGENDS

[0551] FIGS. 1A-1G shows results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 8, 10, 12, 14 and individual tumor growth was monitored to day 41. FIG. 1A and FIG. 1D show results when using IL-2, IL-12sc, and GM-CSF (ModA) mRNA. FIG. 1B and FIG. 1E show IL-2, IL-12sc, and GM-CSF (ModB) mRNA. FIG. 1C and FIG. 1F show results when using luciferase mRNA (ModA). FIG. 1G shows results when using luciferase mRNA (ModB). (N=10 mice/group for A-C and N=9 mice/group for D-G).

[0552] FIGS. 2A-2D show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 19, 21, 24, 26, 28 and 31 and individual tumor growth was monitored to day 48. FIG. 2A shows GM-CSF, IL-2, IL-12sc (ModA). FIG. 2B shows GM-CSF, IL-2, IL-12sc (ModB). FIG. 2C shows luciferase mRNA (ModA) as a control. FIG. 2D shows Ringer's solution as a control.

[0553] FIGS. 3A-3C show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 13, 15, 18, 20 and 22 and tumor growth was monitored to day 42. FIG. 3A shows IL-2, GM-CSF, IL-12sc (ModB). FIG. 3B shows IL-15 sushi, GM-CSF, IL-12sc (ModB). FIG. 3C shows luciferase mRNA (ModB) as a control.

[0554] FIGS. 4A-4F show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45. FIG. 4A shows IL-2, IL-12sc, and GM-CSF (ModA). FIG. 4B is a duplicate in the same experiment as described in FIG. 5A, showing IL-2, IL-12sc, and GM-CSF (ModB). FIG. 4C shows IL-15 sushi, IL-12sc, and GM-CSF (ModA). FIG. 4D is a duplicate in the same experiment as described in FIG. 5B, showing IL-15 sushi, IL-12sc, and GM-CSF (ModB). FIG. 4E shows control luciferase mRNA (ModA). FIG. 4F is a duplicate in the same experiment as described in FIGS. 5D and 6D showing control luciferase mRNA (ModB). (N=8 mice/group).

[0555] FIGS. 5A-5D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45. FIG. 5A is a duplicate in the same experiment as described in FIG. 4B, showing IL-2, IL-12sc, and GM-CSF (ModB). FIG. 5B is a duplicate in the same experiment as described in FIG. 4D, showing IL-15 sushi, IL-12sc, and GM-CSF (ModB). FIG. 5C is a duplicate in the same experiment as described in FIG. 6C, showing IL-2, IL-12sc, GM-CSF, and IFN.alpha. (ModB). FIG. 5D is a duplicate in the same experiment as described in FIGS. 4F and 6D, showing luciferase mRNA (ModB) as control. (N=8 mice/group).

[0556] FIGS. 6A and 6B show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted. FIG. 6A is a duplicate in the same experiment as described in FIG. 7A, showing GM-CSF, IL-2, IL-12sc, IFN.alpha. (ModB). FIG. 6B is a duplicate in the same experiment as described in FIG. 7C, showing luciferase mRNA (ModB). N=8 mice/group.

[0557] FIGS. 6C and 6D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 11, 13, 15, 17 and individual tumor growth was plotted. FIG. 6C is a duplicate in the same experiment as described in FIG. 5C, showing GM-CSF, IL-2, IL-12sc, IFN.alpha. (ModB). FIG. 6D is a duplicate in the same experiment as described in FIGS. 4F and 5D, showing luciferase mRNA (ModB). N=8 mice/group.

[0558] FIGS. 6E and 6F show results of experiments where MC38 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 15, 19, 23 and individual tumor growth was plotted. FIG. 6E shows GM-CSF, IL-2, IL-12sc, IFN.alpha. (ModB). FIG. 6F shows luciferase mRNA (ModB). N=5 mice/group.

[0559] FIGS. 7A-7F show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted. FIG. 7A is a duplicate in the same experiment as described in FIG. 6A, showing IL-2, IL-12sc, GM-CSF, IFN.alpha. (ModB). FIG. 7B shows IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha. (ModB). FIG. 7C is a duplicate in the same experiment as described in FIG. 6B, showing a luciferase mRNA (ModB) control. In a repeat study of similar design, CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 19, 21, 23, 26, 28 and 30 and individual tumor growth was plotted. FIG. 7D is a duplicate of the same experiment as described in FIG. 9A, showing IL-2, IL-12sc, GM-CSF, IFN.alpha. (ModB). FIG. 7E shows IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha. (ModB). FIG. 7F is a duplicate of the same experiment as described in FIG. 9F, showing a luciferase mRNA (ModB) control. N=8 mice/group for Figures A-C and N=10-11 mice/group for Figures D-F.

[0560] FIGS. 8A-8H show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 12, 15, 19 and 22 and individual tumor growth was monitored and plotted to day 35. FIG. 8A shows IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha. (ModB). FIG. 8B shows IL-15 sushi, IL-12sc, IFN.alpha. (ModB). FIG. 8C shows IL-15 sushi, GM-CSF, IFN.alpha. (ModB). FIG. 8D shows GM-CSF, IL-12sc, IFN.alpha. (ModB). FIG. 8E shows IL-15 sushi, GM-CSF, IL-12sc (ModB). FIG. 8F shows a luciferase mRNA (ModB) control. (N=10/group). FIGS. 8G and 8H show tumor growth kinetics of the study shown in FIGS. 8A-8F. FIG. 8G shows mean tumor volumes up to day 33 for all treatment groups. FIG. 8H shows tumor growth repression. T/C (Tumor/Control based on mean tumor volume) was calculated up to day 19.

[0561] FIGS. 9A-9F show experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 19, 21, 23, 26, 28 and 30 and tumor growth was monitored to day 50. FIG. 9A is a duplicate in the same experiment as described in FIG. 7D, showing GM-CSF, IL-2, IL-12sc, IFN.alpha. (ModB). FIG. 9B shows IL-2, IL-12sc, IFN.alpha. (ModB). FIG. 9C shows GM-CSF, IL-2, IFN.alpha. (ModB). FIG. 9D shows GM-CSF, IL-12sc, IFN.alpha. (ModB). FIG. 9E shows GM-CSF, IL-2, IL-12sc (ModB). FIG. 9F is a duplicate in the same experiment as described in FIG. 7F, showing shows luciferase mRNA (ModA) as control. (N=11/group for FIG. 9A-E; luciferase mRNA group N=10 for FIG. 9F).

[0562] FIGS. 10A-10B shows tumor growth kinetics of the study shown in FIG. 9. FIG. 10A shows mean tumor volumes up to day 36 for all treatment groups. FIG. 10B shows tumor growth repression. T/C (Tumor/Control based on mean tumor volume) was calculated up to day 30.

[0563] FIG. 11 shows a bar graph of data from the experiments shown in FIG. 9 showing mRNA mixtures with significant reduction in tumor volume, where the number of mice in each of the treatment groups with significant tumor reduction was compared to the luciferase control group based on Z score of tumor volume and the ratio between tumor volume change and the mean of the control group.

[0564] FIGS. 12A-12D show the results of experiments where mice that were 1) tumor naive, or 2) had been previously injected subcutaneously with 5.times.10.sup.5B16F10 cells and rejected the original tumor following intratumoral cytokine mRNA treatment. Both groups were re-challenged with B16F10 tumors. FIG. 12A shows tumor naive host mice. FIG. 12B shows mice that had previously rejected B16F10 tumors following intratumoral cytokine mRNA treatment with GM-CSF, IL-15sushi, IL-12sc, IFN.alpha. (ModB). Mice were monitored for 55 days following B16F10 injection and tumor growth for each mouse was plotted. All nine naive mice engrafted with B16F10 cells developed tumors (FIG. 12A), whereas all eight tumor-free mice rejected the B16F10 cells and did not exhibit growth of B16F10 tumors (FIG. 12B). The graph in FIG. 12B has no visible data trace because all observations were zero, i.e., overlapping the horizontal axis. FIG. 12C shows an example of localized vitiligo at the tumor site. FIG. 12D shows the results of experiments where mice that were tumor naive (triangle symbol), or had been previously injected subcutaneously with CT26 tumor cells and rejected the original tumor following intratumoral cytokine mRNA treatment (circle symbol). Both groups were re-challenged with either CT26 tumor cells (CT26-WT) or with CT26-.DELTA.gp70 tumor cells, in which the gp70-epitope had been knocked out. Mice were monitored for 21 days following tumor cell injection. All nine but one naive mice engrafted with CT26-WT cells and all naive mice engrafted with CT26-.DELTA.gp70 cells developed tumors, whereas all three tumor-free mice rejected the CT26 tumor cells and did not exhibit growth of CT26 and CT26-.DELTA.gp70 tumors, respectively.

[0565] FIGS. 13A-13D show the results of experiments where mice were implanted with B16F10 tumor cells on day 0 on the right (injected) and left flanks (uninjected) (FIG. 13A). Mice received a series of 4 intratumoral injections with ModB cytokine mRNA (IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.) or ModB control mRNA (luciferase) in the right tumor on days 11, 15, 19, and 23. Mean tumor volumes+/-SEM (n=12) are shown for the injected (FIG. 13B) and the contralateral uninjected tumors (FIG. 13C). Median survival is shown in FIG. 13D.

[0566] FIGS. 14A-14F show results of experiments where human HEK293 (FIG. 14B) and melanoma cell lines (A101D (FIG. 14C), A2058 (FIG. 14D), A375 (FIG. 14E), and Hs294T (FIG. 14F)) were transfected with human cytokine mRNA mixture (IL-12sc, GM-CSF, IL-15 sushi and IFN.alpha.2b) in a range of mRNA doses. Supernatants were collected 24 hrs after transfection and protein concentrations were determined with cytokine specific ELISAs. FIG. 14A shows a schematic of the experiment.

[0567] FIGS. 15A-15B show a schematic (FIG. 15A) and results (FIG. 15B) from a study where a human cytokine mRNA mixture encoding IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b, or individual cytokine mRNAs, were transfected in HEK293 cells and the conditioned media was collected at 24 hrs, diluted and added to human PBMCs. IFN.gamma. was measured in the PBMC culture supernatant at 24 hrs. (N=6 donors, Mean).

[0568] FIGS. 16A-16E show the results of experiments where immune compromised mice bearing human A375 tumor xenografts received a single injection with the ModB mRNA mixture encoding the human cytokines (IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b; "the IL15 sushi mixture") or (IL-2, IL-12sc, GM-CSF and IFN.alpha.2b; "the IL2 mixture"). Tumor cell lysates were prepared at 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, and 72 hrs after injection and the concentration of each cytokine was measured with respect to the total protein in the tumor lysates (n=3 mice/time point, +/-SEM). FIG. 16A shows IFN.alpha.2b, FIG. 16B shows IL-2, FIG. 16C shows IL-12sc, FIG. 16D shows IL-15 sushi, and FIG. 16E shows GM-CSF.

[0569] FIGS. 17A-17C show the results of experiments where mRNA was isolated from A375 tumors at 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, and 72 hrs after injection of ModB cytokine mRNA mixture (IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha.2b) or (IL-2, IL-12sc, GM-CSF, IFN.alpha.2b). Expression of interferon alpha response genes were monitored by qPCR. FIG. 17A shows human ISG15, FIG. 17B shows human ISG54, and FIG. 17C shows human MX1.

[0570] FIGS. 18A-18E show the results of experiments where mice were implanted with B16F10 tumor cells and treated with mRNA mixtures (FLT3L, IL-2, 41BBL, and CD27L-CD40L) with or without IFN.alpha.. mRNA mixtures without IFN.alpha. in standard (ModA, FIG. 18B) and modified forms (ModB, FIG. 18C) were compared to those including IFN.alpha. in standard (ModA, FIG. 18D) and modified forms (ModB, FIG. 18E). FIG. 18A is a negative control where Ringer's media without mRNA was provided.

[0571] FIGS. 19A-19E show the results of experiments where mice were implanted with tumors on one flank and received an IV injection of luciferase-expressing tumor cells that homed to the lung (FIG. 19A). Mice in the treatment group received intratumoral injections of mRNA mixtures IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. into the flank tumor only while tumors in the lung were untreated. FIG. 19B shows exemplarily bioluminescence measurements in lungs and pictures of the according lungs taken out on the same day (day 20); tumor nodes are visual as black marks; FIG. 19C shows mean tumor volume of flank tumors as determined by caliper measurements; FIG. 19D shows total flux analysis of bioluminescence measurements on day 20; FIG. 19E shows lung weights.

[0572] FIGS. 20A-20G show the results of experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic administration of antibodies in dual flank tumor models. Mice implanted with either the B16F10 tumor on the left and right flank or MC38 tumors on the left and right flank received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. (Mod B) into only one flank tumor, while the other flank tumor was untreated. Mice also received intraperitoneal (systemic) injection of an anti-PD1 antibody. FIG. 20 shows overall survival in the B16F10 (FIG. 20A) and MC38 (FIG. 20B) tumor models. FIGS. 20C-G show the results of an experiment evaluating the anti-PD-1 antibody where mice were implanted with B16F10 tumors on one flank and received an IV injection of luciferase-expressing B16F10 tumor cells that homed to the lung. Mice received three intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. and also received three intraperitoneal (systemic) injection of an anti-PD-1 antibody. Tumor growth of the SC tumors is depicted in FIG. 20C-F. FIG. 20C shows control mRNA and control antibody; FIG. 20D shows control mRNA plus anti-PD1 antibody; FIG. 20E shows cytokine mRNA mixture plus isotype control antibody. FIG. 20F shows cytokine mRNA plus anti-PD-1 antibody. FIG. 20G shows percent survival of all four treatment groups until day 70 after IV tumor inoculation; the treatment group that received mRNA plus anti-PD-1 antibody showed strongest anti-tumoral activity with 6 out of 15 mice being tumor-free on day 40 after tumor inoculation.

[0573] FIGS. 21A-21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic administration of antibodies. Mice bearing CT26 tumors received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.. Mice also received intraperitoneal (systemic) injection of an anti-CTLA-4 antibody. FIG. 21A shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 12 out of 16 mice being tumor-free on day 55 after tumor inoculation. FIG. 21B shows cytokine mRNA mixture plus isotype control antibody; FIG. 21C shows control mRNA plus anti-CTLA-4 antibody; FIG. 21D shows control mRNA and control antibody. FIGS. 21E-21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with anti-CTLA-4 antibody in B16F10 tumor model. Mice bearing B16F10 tumors received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.. Mice also received intraperitoneal (systemic) injection of an anti-CTLA-4 antibody. FIG. 21E shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 6 out of 9 mice being tumor-free on day 70 after tumor inoculation. FIG. 21F shows cytokine mRNA mixture plus isotype control antibody; FIG. 21G shows control mRNA plus anti-CTLA-4 antibody; FIG. 21H shows control mRNA and control antibody. FIG. 21I shows percent survival of all four treatment groups until day 70 after tumor inoculation.

[0574] FIGS. 22A-22D shows the results of experiments designed to evaluate the effect of intratumoral injection of cytokine mRNA (Mod B) in human tumor xenografts of different human cancers. Intratumoral expression of each of the 4 mRNA encoded cytokines is shown: IL-12sc (FIG. 22A), IFN.alpha.2b (FIG. 22B), GM-CSF (FIG. 22C), and IL-15 sushi (FIG. 22D).

[0575] FIGS. 23A-23D show the results of experiments designed to evaluate the effect of different intratumoral mRNA doses on the expression of the encoded cytokines: IL-15 sushi (FIG. 23A), IL-12sc (FIG. 23B), GM-CSF (FIG. 23C) and IFN.alpha.2b (FIG. 23D).

[0576] FIGS. 24A-24G show the results of experiments where mice were implanted with B16F10 tumor, and treated with four intratumoral injections of cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha. (ModB) or mRNA encoding a single cytokine. Tumor volume out to approximately day 70 was measured. FIG. 24A shows luciferase control; FIG. 24B shows the four-cytokine mixture; FIG. 24C shows IL-12sc mRNA only; FIG. 24D shows GM-CSF mRNA only; FIG. 24E shows IFN.alpha. mRNA only; and FIG. 24F shows IL-15 sushi only. FIG. 24G shows overall survival of B16F10 tumors treated with cytokine mRNA mixture or individual mRNA encoded cytokines. Survival data is from experiment presented in FIG. 24A-F.

[0577] FIG. 25 shows CD8+ immune cell infiltrate in subcutaneous tumors after control mRNA ("placebo") and cytokine mRNA treatment.

[0578] FIGS. 26A-26C show results of measurements of CD8+ T cells specific for the gp70 tumor antigen of gp70 in blood of CT26 tumor bearing mice that had received intratumoral administration of cytokine mRNA treatment and control mRNA, respectively. FIG. 26C shows exemplarily a FACS histogram of CD8+ T cells stained with anti-mouse CD8 antibody and with the gp70-specific tetramer derived from an animal that had received control mRNA and FIG. 26B shows the example from one animal treated with cytokine mRNA. FIG. 26C shows the analysis of percentage of gp70-specific CD8+ T-cells in blood 13 days after treatment start from 9 mice that had received four injections of control mRNA and 10 mice that had received 4 injections of cytokine mRNA.

[0579] FIGS. 27A-27C show experiments where mice bearing B16F10 tumors on the left and right flanks received a single intratumoral mRNA injection with cytokine mRNA or control mRNA in only one tumor. On day 7 following the mRNA injection the left and right tumors were collected and subjected to RNA sequencing. FIG. 27B shows the results of ingenuity pathway analysis comparing the gene expression changes between the cytokine mRNA treatment vs control mRNA treated tumors. Causal network analysis for treated tumor side (Column 1) and untreated tumor side (Column 2) was performed and Activation Z score (Top half) and Inhibition Z score (Lower half) was analyzed to define pathways up and down regulated, respectively. FIG. 27 shows cluster analysis of injected and non-injected tumors was performed based on 327 interferon gamma regulated genes. Both the injected and non-injected tumors of mice treated with cytokine mRNA showed upregulation of multiple IFN gamma genes in comparison to mice treated with control mRNA.

[0580] FIGS. 28A-28D show fluorescence micrographs of cells from a B16F10 dualtumor model. Panel A shows the injected tumor treated with cytokine mRNA and panel B shows the corresponding uninjected tumor. Panel C shows the injected tumor treated with control mRNA and panel D shows the corresponding uninjected tumor. The slides were stained for CD4+, CD8+, and FoxP3+ cells.

[0581] FIGS. 28E-G show frequency of CD4+, CD8+ and FOXP3+ cells quantified in the immunofluorescent images. The frequency of CD4+ and CD8+ cells/mm.sup.2 is presented in FIGS. 28E and 28F. The ratio of the CD8+ frequency divided by FOXP3+ frequency is presented in FIG. 28G.

[0582] FIGS. 29A-29G show mice with a single B16F10 tumor received a single injection with either mRNA encoding the Thy1.1 cell surface protein or vehicle alone (Ringer's solution). At approximately 16-18 hours following intratumoral injection the tumor was excised, digested, stained with a panel of antibodies and analyzed by flow cytometry. The cell type and frequency of cells expressing Thy1.1 were characterized.

[0583] FIGS. 30A-30F show expression of the indicated proteins following various doses of cytokine mRNA or luciferase control mRNA detected in tumor lysates as described in Example 15. "IFNy" in FIG. 30E indicates IFN.gamma..

[0584] FIGS. 31A-31B show flow cytometry results for CD8+ and FOXP3+(Treg) cells following control or cytokine mRNA treatments as described in Example 15. The observed ratio of CD8+ to Treg cells is shown in each panel.

[0585] FIGS. 31C-31D show flow cytometry results for polyfunctional CD8+ T cells following control or cytokine mRNA treatments as described in Example 15. The proportion of polyfunctional CD8+ T cells is shown in each panel.

[0586] FIG. 31E shows the level of PD-L1 on infiltrating myeloid cells following control or cytokine mRNA treatments as described in Example 15.

[0587] FIG. 31F shows the level of PD-1 on infiltrating CD8+ cells following control or cytokine mRNA treatments as described in Example 15.

[0588] FIGS. 31G-H show the frequency of intratumoral Granzyme B CD8+ T cells following control or cytokine mRNA treatments as described in Example 15.

[0589] FIGS. 32A-32B show luciferase expression in various tissues following intratumoral injection of 50 .mu.g mRNA encoding firefly luciferase as described in Example 16.

[0590] FIG. 33 shows data relating to an experiment essentially as shown in FIG. 12.

[0591] FIG. 34 shows the effect of depleting CD8+ T cells, CD4+ T cells or NK cells before treatment with cytokine mRNAs on survival in mice bearing B16F10 tumors as described in Example 17.

[0592] FIG. 35 shows survival of WT and IFN.gamma. KO mice implanted with B16F10 tumor cells as described in Example 1 and treated with control or cytokine mRNAs as described in Example 18.

[0593] FIG. 36 shows a "peri-tumorally," or "peri-tumoral," area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery. The peri-tumoral area comprises host tissue.

DESCRIPTION OF THE SEQUENCES

[0594] Tables 1 and 2 provide a listing of certain sequences referenced herein.

TABLE-US-00001 TABLE 1 DESCRIPTION OF THE SEQUENCES (human sequences) SEQ ID NO: Description SEQUENCE 5' UTR 1 ModA 5' UTR GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (DNA) 2 ModA 5' UTR GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (RNA) 3 ModB 5' UTR GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA (DNA) TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCC 4 ModB 5' UTR GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCC 5 Alternative AGACGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC Mod 5' UTR (DNA) 6 Alternative AGACGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Mod 5' UTR (RNA) 3' UTR 7 ModA/B 3' CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGAC- CTCGGGTCCCAGGTA UTR (DNA) TGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGC- AGCAATGCAGCTCAAAACGCTTAGC CTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT- AACCCCAGGGTTGGTC AATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCTAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAGCATA TGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAA 8 ModA/B 3' CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGAC- CUCGGGUCCCAGGUA UTR (RNA) UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGC- AGCAAUGCAGCUCAAAACGCUUAGC CUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACU- AACCCCAGGGUUGGUC AAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCUAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAGCAUA UGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAA IL-2 9 Human IL-2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEL- KHLQCLEEELKPLEE (amino VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT acid) 10 Human non- ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAA- GTTCTACAAAGAAAA optimized CACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATT- ACAAGAATCCCAAACTCACCAGGAT IL-2 (CDS GCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGA- AGAAGAACTCAAACCTCTGGAGGAA DNA) GTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGT- AATAGTTCTGGAACTAAAGG GATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGA- TTACCTTTTGTCAAAG CATCATCTCAACACTGACTTGATGA 11 Human ATGTACAGAATGCAGCTGCTGTCTTGCATTGCTCTTTCTCTTGCTCTTGTGACAAATTCTGCTCC- AACATCTTCTTCAACAAAGAAAA optimized CACAGCTTCAGCTTGAACACCTTCTTCTTGATCTTCAGATGATTCTGAATGGAATCAACAATT- ACAAAAATCCAAAACTGACAAGAAT IL-2 (CDS GCTGACATTTAAATTTTACATGCCAAAGAAAGCAACAGAACTGAAACACCTTCAGTGCCTTGA- AGAAGAACTGAAACCTCTGGAAGAA DNA) GTGCTGAATCTGGCTCAGAGCAAAAATTTTCACCTGAGACCAAGAGATCTGATCAGCAACATCAATGT- GATTGTGCTGGAACTGAAAG GATCTGAAACAACATTCATGTGTGAATATGCTGATGAAACAGCAACAATTGTGGAATTTCTGAACAGATGGA- TCACATTTTGCCAGTC AATCATTTCAACACTGACATGATGA 12 Human non- AUGUACAGGAUGCAACUCCUGUCUUGCAUUGCACUAAGUCUUGCACUUGUCACAAACAGUGCACCUACUUCAA- GUUCUACAAAGAAAA optimized CACAGCUACAACUGGAGCAUUUACUGCUGGAUUUACAGAUGAUUUUGAAUGGAAUUAAUAAUU- ACAAGAAUCCCAAACUCACCAGGAU IL-2 (RNA GCUCACAUUUAAGUUUUACAUGCCCAAGAAGGCCACAGAACUGAAACAUCUUCAGUGUCUAGA- AGAAGAACUCAAACCUCUGGAGGAA encoding GUGCUAAAUUUAGCUCAAAGCAAAAACUUUCACUUAAGACCCAGGGACUUAAUCAGCAAUAUCA- ACGUAAUAGUUCUGGAACUAAAGG CDS) GAUCUGAAACAACAUUCAUGUGUGAAUAUGCUGAUGAGACAGCAACCAUUGUAGAAUUUCUGAACAGA- UGGAUUACCUUUUGUCAAAG CAUCAUCUCAACACUGACUUGAUGA 13 Human AUGUACAGAAUGCAGCUGCUGUCUUGCAUUGCUCUUUCUCUUGCUCUUGUGACAAAUUCUGCUCC- AACAUCUUCUUCAACAAAGAAAA optimized CACAGCUUCAGCUUGAACACCUUCUUCUUGAUCUUCAGAUGAUUCUGAAUGGAAUCAACAAUU- ACAAAAAUCCAAAACUGACAAGAAU IL-2 (RNA GCUGACAUUUAAAUUUUACAUGCCAAAGAAAGCAACAGAACUGAAACACCUUCAGUGCCUUGA- AGAAGAACUGAAACCUCUGGAAGAA encoding GUGCUGAAUCUGGCUCAGAGCAAAAAUUUUCACCUGAGACCAAGAGAUCUGAUCAGCAACAUCA- AUGUGAUUGUGCUGGAACUGAAAG CDS) GAUCUGAAACAACAUUCAUGUGUGAAUAUGCUGAUGAAACAGCAACAAUUGUGGAAUUUCUGAACAGA- UGGAUCACAUUUUGCCAGUC AAUCAUUUCAACACUGACAUGAUGA IL-12sc 14 Human IL- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGK- TLTIQVKEFGDAGQY 12sc (amino TCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRG- SSDPQGVTCGAATLS acid) AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQL- KPLKNSRQVEVSWEYPDTWST PHSYFSLTFCVQVQGKSKREKKDRVETDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGSSGGGGS- PGGGSSRNLPVATPDP GMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSF- ITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTK- IKLCILLHAFRIRAVT IDRVMSYLNAS 15 Human non- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGG- AACTGAAGAAAGATG optimized TTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTG- ACACCCCTGAAGAAGATGGTATCAC IL-12sc CTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAG- AGTTTGGAGATGCTGGCCAGTAC (CDS DNA) ACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGAT- GGAATTTGGTCCACTGATATTTTAA Sequence AGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACG- TTTCACCTGCTGGTGGCTGACGAC annotation AATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGGTCTTCTGACCCCCAAGGGGTGACGTGCGGA- GCTGCTACACTCTCT CAPS: p40 GCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT- GCCTGCCCAGCTGCTGAGGAGAGTC domain; TGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTC- TTCATCAGGGACATCATCAAACC CAPS: TGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAG- TACCCTGACACCTGGAGTACT linker; CCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAA- AGATAGAGTCTTCACGGACAAGA CAPS: p35. CCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATC- TTGGAGCGAATGGGC ATCTGTGCCCTGCAGTGGCTCTAGCGGAGGGGGAGGCTCTCCTGGCGGGGGATCTAGCAGAAACCTCCCCGT- GGCCACTCCAGACCCA GGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGA- CAAACTCTAGAATTTT ACCCTTGCACTTCTGAGGAAATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTT- TACCATTGGAATTAAC CAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAA- GACCTCTTTTATGATG GCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAG- CTTCTGATGGATCCTA AGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACA- GTGAGACTGTGCCACA AAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAG- AATTCGGGCAGTGACT ATTGATAGAGTGATGAGCTATCTGAATGCTTCCTGATGA 16 Human ATGTGTCACCAGCAGCTGGTGATCTCATGGTTCTCCCTGGTATTTCTGGCATCTCCTCTTGTCGC- AATCTGGGAACTGAAGAAAGACG optimized TGTATGTCGTTGAGCTCGACTGGTATCCGGATGCGCCTGGCGAGATGGTGGTGCTGACCTGTG- ACACCCCAGAGGAGGATGGGATCAC IL-12sc TTGGACCCTTGATCAATCCTCCGAAGTGCTCGGGTCTGGCAAGACTCTGACCATACAAGTGAAAG- AGTTTGGCGATGCCGGGCAGTAC (CDS DNA) ACTTGCCATAAGGGCGGAGAAGTTCTGTCCCACTCACTGCTGCTGCTGCACAAGAAAGAGGAC- GGAATTTGGAGTACCGATATCCTGA Sequence AAGATCAGAAAGAGCCCAAGAACAAAACCTTCTTGCGGTGCGAAGCCAAGAACTACTCAGGGAG- ATTTACTTGTTGGTGGCTGACGAC annotation GATCAGCACCGATCTGACTTTCTCCGTGAAATCAAGTAGGGGATCATCTGACCCTCAAGGAGTCACATGTGGA- GCGGCTACTCTGAGC CAPS: p40 GCTGAACGCGTAAGAGGGGACAATAAGGAGTACGAGTATAGCGTTGAGTGCCAAGAGGATAGC- GCATGCCCCGCCGCCGAAGAATCAT domain; TGCCCATTGAAGTGATGGTGGATGCTGTACACAAGCTGAAGTATGAGAACTACACAAGCTCCTTC- TTCATCCGTGACATCATCAAACC CAPS: AGATCCTCCTAAGAACCTCCAGCTTAAACCTCTGAAGAACTCTAGACAGGTGGAAGTGTCTTGGGAG- TATCCCGACACCTGGTCTACA linker; CCACATTCCTACTTCAGTCTCACATTCTGCGTTCAGGTACAGGGCAAGTCCAAAAGGGAGAAGAA- GGATCGGGTCTTTACAGATAAAA CAPS: p35. CAAGTGCCACCGTTATATGCCGGAAGAATGCCTCTATTTCTGTGCGTGCGCAGGACAGATACTATAGCAGCTC- TTGGAGTGAATGGGC CAGTGTCCCATGTTCAGGGTCATCCGGTGGTGGCGGCAGCCCCGGAGGCGGTAGCTCCAGAAATCTCCCTGT- GGCTACACCTGATCCA GGCATGTTTCCCTGTTTGCACCATAGCCAAAACCTCCTGAGAGCAGTCAGCAACATGCTCCAGAAAGCTAGA- CAAACACTGGAATTCT ACCCATGCACCTCCGAGGAAATAGATCACGAGGATATCACTAAGGACAAAACAAGCACTGTCGAAGCATGCC- TTCCCTTGGAACTGAC AAAGAACGAGAGTTGCCTTAATTCAAGAGAAACATCTTTCATTACAAACGGTAGCTGCTTGGCAAGCAGAAA- AACATCTTTTATGATG GCCCTTTGTCTGAGCAGTATTTATGAGGATCTCAAAATGTACCAGGTGGAGTTTAAGACCATGAATGCCAAG- CTGCTGATGGACCCAA AGAGACAGATTTTCCTCGATCAGAATATGCTGGCTGTGATTGATGAACTGATGCAGGCCTTGAATTTCAACA- GCGAAACCGTTCCCCA GAAAAGCAGTCTTGAAGAACCTGACTTTTATAAGACCAAGATCAAACTGTGTATTCTCCTGCATGCCTTTAG- AATCAGAGCAGTCACT ATAGATAGAGTGATGTCCTACCTGAATGCTTCCTGATGA 17 Human non- AUGUGUCACCAGCAGUUGGUCAUCUCUUGGUUUUCCCUGGUUUUUCUGGCAUCUCCCCUCGUGGCCAUAUGGG- AACUGAAGAAAGAUG optimized UUUAUGUCGUAGAAUUGGAUUGGUAUCCGGAUGCCCCUGGAGAAAUGGUGGUCCUCACCUGUG- ACACCCCUGAAGAAGAUGGUAUCAC IL-12sc CUGGACCUUGGACCAGAGCAGUGAGGUCUUAGGCUCUGGCAAAACCCUGACCAUCCAAGUCAAAG- AGUUUGGAGAUGCUGGCCAGUAC (RNA ACCUGUCACAAAGGAGGCGAGGUUCUAAGCCAUUCGCUCCUGCUGCUUCACAAAAAGGAAGAUGGAAU- UUGGUCCACUGAUAUUUUAA encoding AGGACCAGAAAGAACCCAAAAAUAAGACCUUUCUAAGAUGCGAGGCCAAGAAUUAUUCUGGACG- UUUCACCUGCUGGUGGCUGACGAC CDS) AAUCAGUACUGAUUUGACAUUCAGUGUCAAAAGCAGCAGAGGGUCUUCUGACCCCCAAGGGGUGACGU- GCGGAGCUGCUACACUCUCU GCAGAGAGAGUCAGAGGGGACAACAAGGAGUAUGAGUACUCAGUGGAGUGCCAGGAGGACAGUGCCUGCCCA- GCUGCUGAGGAGAGUC UGCCCAUUGAGGUCAUGGUGGAUGCCGUUCACAAGCUCAAGUAUGAAAACUACACCAGCAGCUUCUUCAUCA- GGGACAUCAUCAAACC UGACCCACCCAAGAACUUGCAGCUGAAGCCAUUAAAGAAUUCUCGGCAGGUGGAGGUCAGCUGGGAGUACCC- UGACACCUGGAGUACU CCACAUUCCUACUUCUCCCUGACAUUCUGCGUUCAGGUCCAGGGCAAGAGCAAGAGAGAAAAGAAAGAUAGA- GUCUUCACGGACAAGA CCUCAGCCACGGUCAUCUGCCGCAAAAAUGCCAGCAUUAGCGUGCGGGCCCAGGACCGCUACUAUAGCUCAU- CUUGGAGCGAAUGGGC AUCUGUGCCCUGCAGUGGCUCUAGCGGAGGGGGAGGCUCUCCUGGCGGGGGAUCUAGCAGAAACCUCCCCGU- GGCCACUCCAGACCCA GGAAUGUUCCCAUGCCUUCACCACUCCCAAAACCUGCUGAGGGCCGUCAGCAACAUGCUCCAGAAGGCCAGA- CAAACUCUAGAAUUUU ACCCUUGCACUUCUGAGGAAAUUGAUCAUGAAGAUAUCACAAAAGAUAAAACCAGCACAGUGGAGGCCUGUU- UACCAUUGGAAUUAAC CAAGAAUGAGAGUUGCCUAAAUUCCAGAGAGACCUCUUUCAUAACUAAUGGGAGUUGCCUGGCCUCCAGAAA- GACCUCUUUUAUGAUG GCCCUGUGCCUUAGUAGUAUUUAUGAAGACUUGAAGAUGUACCAGGUGGAGUUCAAGACCAUGAAUGCAAAG- CUUCUGAUGGAUCCUA AGAGGCAGAUCUUUCUAGAUCAAAACAUGCUGGCAGUUAUUGAUGAGCUGAUGCAGGCCCUGAAUUUCAACA- GUGAGACUGUGCCACA AAAAUCCUCCCUUGAAGAACCGGAUUUUUAUAAAACUAAAAUCAAGCUCUGCAUACUUCUUCAUGCUUUCAG- AAUUCGGGCAGUGACU AUUGAUAGAGUGAUGAGCUAUCUGAAUGCUUCCUGAUGA

18 Human AUGUGUCACCAGCAGCUGGUGAUCUCAUGGUUCUCCCUGGUAUUUCUGGCAUCUCCUCUUGUCGC- AAUCUGGGAACUGAAGAAAGACG Optimized UGUAUGUCGUUGAGCUCGACUGGUAUCCGGAUGCGCCUGGCGAGAUGGUGGUGCUGACCUGUG- ACACCCCAGAGGAGGAUGGGAUCAC IL-12sc UUGGACCCUUGAUCAAUCCUCCGAAGUGCUCGGGUCUGGCAAGACUCUGACCAUACAAGUGAAAG- AGUUUGGCGAUGCCGGGCAGUAC (RNA ACUUGCCAUAAGGGCGGAGAAGUUCUGUCCCACUCACUGCUGCUGCUGCACAAGAAAGAGGACGGAAU- UUGGAGUACCGAUAUCCUGA encoding AAGAUCAGAAAGAGCCCAAGAACAAAACCUUCUUGCGGUGCGAAGCCAAGAACUACUCAGGGAG- AUUUACUUGUUGGUGGCUGACGAC CDS) GAUCAGCACCGAUCUGACUUUCUCCGUGAAAUCAAGUAGGGGAUCAUCUGACCCUCAAGGAGUCACAU- GUGGAGCGGCUACUCUGAGC GCUGAACGCGUAAGAGGGGACAAUAAGGAGUACGAGUAUAGCGUUGAGUGCCAAGAGGAUAGCGCAUGCCCC- GCCGCCGAAGAAUCAU UGCCCAUUGAAGUGAUGGUGGAUGCUGUACACAAGCUGAAGUAUGAGAACUACACAAGCUCCUUCUUCAUCC- GUGACAUCAUCAAACC AGAUCCUCCUAAGAACCUCCAGCUUAAACCUCUGAAGAACUCUAGACAGGUGGAAGUGUCUUGGGAGUAUCC- CGACACCUGGUCUACA CCACAUUCCUACUUCAGUCUCACAUUCUGCGUUCAGGUACAGGGCAAGUCCAAAAGGGAGAAGAAGGAUCGG- GUCUUUACAGAUAAAA CAAGUGCCACCGUUAUAUGCCGGAAGAAUGCCUCUAUUUCUGUGCGUGCGCAGGACAGAUACUAUAGCAGCU- CUUGGAGUGAAUGGGC CAGUGUCCCAUGUUCAGGGUCAUCCGGUGGUGGCGGCAGCCCCGGAGGCGGUAGCUCCAGAAAUCUCCCUGU- GGCUACACCUGAUCCA GGCAUGUUUCCCUGUUUGCACCAUAGCCAAAACCUCCUGAGAGCAGUCAGCAACAUGCUCCAGAAAGCUAGA- CAAACACUGGAAUUCU ACCCAUGCACCUCCGAGGAAAUAGAUCACGAGGAUAUCACUAAGGACAAAACAAGCACUGUCGAAGCAUGCC- UUCCCUUGGAACUGAC AAAGAACGAGAGUUGCCUUAAUUCAAGAGAAACAUCUUUCAUUACAAACGGUAGCUGCUUGGCAAGCAGAAA- AACAUCUUUUAUGAUG GCCCUUUGUCUGAGCAGUAUUUAUGAGGAUCUCAAAAUGUACCAGGUGGAGUUUAAGACCAUGAAUGCCAAG- CUGCUGAUGGACCCAA AGAGACAGAUUUUCCUCGAUCAGAAUAUGCUGGCUGUGAUUGAUGAACUGAUGCAGGCCUUGAAUUUCAACA- GCGAAACCGUUCCCCA GAAAAGCAGUCUUGAAGAACCUGACUUUUAUAAGACCAAGAUCAAACUGUGUAUUCUCCUGCAUGCCUUUAG- AAUCAGAGCAGUCACU AUAGAUAGAGUGAUGUCCUACCUGAAUGCUUCCUGAUGA IFNalpha2b (IFN.alpha.2b) 19 Human MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE- FGNQFQKAETIPVLHEMIQQIFN IFN.alpha.2b LFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPC- AWEVVRAEIMRSFSL (amino STNLQESLRSKE acid) 20 Human non- ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGCTGTG- ATCTGCCTCAAACCC optimized ACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCT- CCTGCTTGAAGGACAGACATGACTT IFN.alpha.2b TGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATC- CAGCAGATCTTCAAC (CDS DNA) CTTTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACT- GAACTCTACCAGCAGCTGAATGACC TGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTG- TGAGGAAATACTTCCA AAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCAT- GAGATCTTTTTCTTTG TCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGATGA 21 Human ATGGCCCTGACTTTTGCCCTTCTCGTGGCTTTGTTGGTGCTGAGTTGCAAATCTTCCTGTAGTGT- CGGATGTGATCTGCCTCAAACCC optimized ACAGTCTGGGATCTAGGAGAACACTGATGCTGTTGGCACAGATGAGGAGAATTAGCCTCTTTT- CCTGCCTGAAGGATAGACATGACTT IFN.alpha.2b CGGCTTTCCCCAAGAGGAGTTTGGCAATCAGTTCCAGAAAGCGGAAACGATTCCCGTTCTGCACGAGATGATC- CAGCAGATCTTCAAC (CDS DNA) CTCTTTTCAACCAAAGACAGCTCAGCAGCCTGGGATGAGACACTGCTGGACAAATTCTACACA- GAACTGTATCAGCAGCTTAACGATC TGGAGGCATGCGTGATCCAAGGGGTTGGTGTGACTGAAACTCCGCTTATGAAGGAGGACTCCATTCTGGCTG- TACGGAAGTACTTCCA GAGAATAACCCTCTATCTGAAGGAGAAGAAGTACTCACCATGTGCTTGGGAAGTCGTGAGAGCCGAAATCAT- GAGATCCTTCAGCCTT AGCACCAATCTCCAGGAATCTCTGAGAAGCAAAGAGTGATGA 22 Human non- AUGGCCUUGACCUUUGCUUUACUGGUGGCCCUCCUGGUGCUCAGCUGCAAGUCAAGCUGCUCUGUGGGCUGUG- AUCUGCCUCAAACCC optimized ACAGCCUGGGUAGCAGGAGGACCUUGAUGCUCCUGGCACAGAUGAGGAGAAUCUCUCUUUUCU- CCUGCUUGAAGGACAGACAUGACUU IFN.alpha.2b (RNA UGGAUUUCCCCAGGAGGAGUUUGGCAACCAGUUCCAAAAGGCUGAAACCAUCCCUGUCCUCCAUGAGAUGAUC- CAGCAGAUCUUCAAC encoding CUUUUCAGCACAAAGGACUCAUCUGCUGCUUGGGAUGAGACCCUCCUAGACAAAUUCUACACUG- AACUCUACCAGCAGCUGAAUGACC CDS) UGGAAGCCUGUGUGAUACAGGGGGUGGGGGUGACAGAGACUCCCCUGAUGAAGGAGGACUCCAUUCUG- GCUGUGAGGAAAUACUUCCA AAGAAUCACUCUCUAUCUGAAAGAGAAGAAAUACAGCCCUUGUGCCUGGGAGGUUGUCAGAGCAGAAAUCAU- GAGAUCUUUUUCUUUG UCAACAAACUUGCAAGAAAGUUUAAGAAGUAAGGAAUGAUGA 23 Human AUGGCCCUGACUUUUGCCCUUCUCGUGGCUUUGUUGGUGCUGAGUUGCAAAUCUUCCUGUAGUGU- CGGAUGUGAUCUGCCUCAAACCC optimized ACAGUCUGGGAUCUAGGAGAACACUGAUGCUGUUGGCACAGAUGAGGAGAAUUAGCCUCUUUU- CCUGCCUGAAGGAUAGACAUGACUU IFN.alpha.2b (RNA CGGCUUUCCCCAAGAGGAGUUUGGCAAUCAGUUCCAGAAAGCGGAAACGAUUCCCGUUCUGCACGAGAUGAUC- CAGCAGAUCUUCAAC encoding CUCUUUUCAACCAAAGACAGCUCAGCAGCCUGGGAUGAGACACUGCUGGACAAAUUCUACACAG- AACUGUAUCAGCAGCUUAACGAUC CDS) UGGAGGCAUGCGUGAUCCAAGGGGUUGGUGUGACUGAAACUCCGCUUAUGAAGGAGGACUCCAUUCUG- GCUGUACGGAAGUACUUCCA GAGAAUAACCCUCUAUCUGAAGGAGAAGAAGUACUCACCAUGUGCUUGGGAAGUCGUGAGAGCCGAAAUCAU- GAGAUCCUUCAGCCUU AGCACCAAUCUCCAGGAAUCUCUGAGAAGCAAAGAGUGAUGA IL-15 sushi 24 Human IL-15 MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT- ECVLNKATNVAHWTT sushi PSLKCIRDPALVHQRPAPPGGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTE- SDVHPSCKVTAMKCFLLELQV (amino ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECKELEEKNIKEFLQSFVHIVQMFINTS acid) 25 Human IL-15 ATGGCCCCGCGGCGGGCGCGCGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTGCTGCTGCTCCGGC- CGCCGGCGACGCGGG sushi GCATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA- CTCCAGGGAGCGGTACATTTG (CDS DNA) TAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGC- CACGAATGTCGCCCACTGGACAACC Sequence CCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCCACCCGGGGGAG- GATCTGGCGGCGGTGGGTCTGGCG annotations GGGGATCTGGCGGAGGAGGAAGCTTACAGAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCT- TATTCAATCTATGCA CAPS: IL-15 TATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTC- TTGGAGTTACAAGTT sushi; ATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAAC- AACAGTTTGTCTTCTAATGGGA CAPS: ATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCA- GAGTTTTGTACATATTGTCCA linker; AATGTTCATCAACACTTCTTGATGA CAPS: mature IL-15 26 Human IL-15 AUGGCCCCGCGGCGGGCGCGCGGCUGCCGGACCCUCGGUCUCCCGGCGCUGCUACUGCUGCUGCUGCUCCGGC- CGCCGGCGACGCGGG sushi (RNA GCAUCACGUGCCCUCCCCCCAUGUCCGUGGAACACGCAGACAUCUGGGUCAAGAGCUACAGCUUGUACUCCAG- GGAGCGGUACAUUUG encoding UAACUCUGGUUUCAAGCGUAAAGCCGGCACGUCCAGCCUGACGGAGUGCGUGUUGAACAAGGCC- ACGAAUGUCGCCCACUGGACAACC CDS) CCCAGUCUCAAAUGCAUUAGAGACCCUGCCCUGGUUCACCAAAGGCCAGCGCCACCCGGGGGAGGAUC- UGGCGGCGGUGGGUCUGGCG GGGGAUCUGGCGGAGGAGGAAGCUUACAGAACUGGGUGAAUGUAAUAAGUGAUUUGAAAAAAAUUGAAGAUC- UUAUUCAAUCUAUGCA UAUUGAUGCUACUUUAUAUACGGAAAGUGAUGUUCACCCCAGUUGCAAAGUAACAGCAAUGAAGUGCUUUCU- CUUGGAGUUACAAGUU AUUUCACUUGAGUCCGGAGAUGCAAGUAUUCAUGAUACAGUAGAAAAUCUGAUCAUCCUAGCAAACAACAGU- UUGUCUUCUAAUGGGA AUGUAACAGAAUCUGGAUGCAAAGAAUGUGAGGAACUGGAGGAAAAAAAUAUUAAAGAAUUUUUGCAGAGUU- UUGUACAUAUUGUCCA AAUGUUCAUCAACACUUCUUGAUGA GM-CSF 27 Human GM- MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQ- TRLELYKQGLRGSLT CSF (amino KLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE acid) 28 Human GM- ATGTGGCTCCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCTCCATCTCTGCACCCGCCCGCTCGCCCAGCC- CCAGCACGCAGCCCT CSF GGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTGCTGAACCTGAGTAGAGACACTGCTGCTGAGA- TGAATGAAACAGTAGAAGT (CDS DNA) CATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAA- GCAGGGCCTGCGGGGCAGCCTCACC AAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCC- TGTGCAACCCAGATTA TCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAG- TCCAGGAGTGATGA 29 Human GM- AUGUGGCUCCAGAGCCUGCUGCUCUUGGGCACUGUGGCCUGCUCCAUCUCUGCACCCGCCCGCUCGCCCAGCC- CCAGCACGCAGCCCU CSF (RNA GGGAGCAUGUGAAUGCCAUCCAGGAGGCCCGGCGUCUGCUGAACCUGAGUAGAGACACUGCUGC- UGAGAUGAAUGAAACAGUAGAAGU encoding CAUCUCAGAAAUGUUUGACCUCCAGGAGCCGACCUGCCUACAGACCCGCCUGGAGCUGUACAAG- CAGGGCCUGCGGGGCAGCCUCACC CDS) AAGCUCAAGGGCCCCUUGACCAUGAUGGCCAGCCACUACAAGCAGCACUGCCCUCCAACCCCGGAAAC- UUCCUGUGCAACCCAGAUUA UCACCUUUGAAAGUUUCAAAGAGAACCUGAAGGACUUUCUGCUUGUCAUCCCCUUUGACUGCUGGGAGCCAG- UCCAGGAGUGAUGA

TABLE-US-00002 TABLE 2 DESCRIPTION OF THE SEQUENCES (mouse sequences and other sequences of the invention) SEQ ID NO: Description SEQUENCE IL-2 mouse 30 ModA IL-2 MRVTAPRTLILLLSGALALTETWAGSGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY- MPKKATELKHLQCLE (amino EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ- SIISTLT acid, human IL-2 in combination with a mouse optimized secretion sequence) 31 ModA IL-2 GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGACCGCCCCCAGA- ACCCTGATCCTGCTG (DNA: CTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGCGGATCCGCACCTACTTCAAGTTCTA- CAAAGAAAACACAGCTACAAC 5'UTR- TGGAGCATTTACTTCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAAC- TCACCAGGATGCTCACATTTAA CDS-3'UTR) GTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAG- GAAGTGCTAAATTTA GCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTA- AAGGGATCTGAAACAA CATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTC- AAAGCATCATCTCAAC ACTGACTTGACTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCA- ACTACTAAACTGGGGG ATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGAGAG- CTCGCTTTCTTGCTGT CCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTG- AGCATCTGGATTCTGC CTAATAAAAAACATTTATTTTCATTGCTGCGTCGAGACCTGGTCCAGAGTCGCTAGCAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAG CATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAA 32 ModA IL-2 GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGACCGCCCCCAGA- ACCCUGAUCCUGCUG (RNA) CUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGCGGAUCCGCACCUACUUCAAGUUCUA- CAAAGAAAACACAGCUACAAC UGGAGCAUUUACUUCUGGAUUUACAGAUGAUUUUGAAUGGAAUUAAUAAUUACAAGAAUCCCAAACUCACCA- GGAUGCUCACAUUUAA GUUUUACAUGCCCAAGAAGGCCACAGAACUGAAACAUCUUCAGUGUCUAGAAGAAGAACUCAAACCUCUGGA- GGAAGUGCUAAAUUUA GCUCAAAGCAAAAACUUUCACUUAAGACCCAGGGACUUAAUCAGCAAUAUCAACGUAAUAGUUCUGGAACUA- AAGGGAUCUGAAACAA CAUUCAUGUGUGAAUAUGCUGAUGAGACAGCAACCAUUGUAGAAUUUCUGAACAGAUGGAUUACCUUUUGUC- AAAGCAUCAUCUCAAC ACUGACUUGACUCGAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCA- ACUACUAAACUGGGGG AUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAGAG- CUCGCUUUCUUGCUGU CCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUG- AGCAUCUGGAUUCUGC CUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAGACCUGGUCCAGAGUCGCUAGCAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAG CAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAA 33 ModB IL-2 MGAMAPRTLLLLLAAALAPTQTRAGPGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY- MPKKATELKHLQCLE (amino EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ- SIISTLT acid) 34 ModB IL-2 GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA (DNA: TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCA- TGGCCCCTAGAACATTGCTCC 5'UTR- TGCTGCTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCGCACCTACTTCAA- GTTCTACAAAGAAAACACAGCT CDS-3'UTR) ACAACTGGAGCATTTACTTCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTC- ACCAGGATGCTCACA TTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCT- CTGGAGGAAGTGCTAA ATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGG- AACTAAAGGGATCTGA AACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTT- TTGTCAAAGCATCATC TCAACACTGACTTGACTCGACGTCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGT- ACCCCGAGTCTCCCCC GACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC- CAAGCACGCAGCAATG CAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAA- GTTTAACTAAGCTATA CTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAGCATATGAC TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 35 ModB IL-2 GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCA- UGGCCCCUAGAACAUUGCUCC UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCGCACCUACUUCAAGUUCUA- CAAAGAAAACACAGCU ACAACUGGAGCAUUUACUUCUGGAUUUACAGAUGAUUUUGAAUGGAAUUAAUAAUUACAAGAAUCCCAAACU- CACCAGGAUGCUCACA UUUAAGUUUUACAUGCCCAAGAAGGCCACAGAACUGAAACAUCUUCAGUGUCUAGAAGAAGAACUCAAACCU- CUGGAGGAAGUGCUAA AUUUAGCUCAAAGCAAAAACUUUCACUUAAGACCCAGGGACUUAAUCAGCAAUAUCAACGUAAUAGUUCUGG- AACUAAAGGGAUCUGA AACAACAUUCAUGUGUGAAUAUGCUGAUGAGACAGCAACCAUUGUAGAAUUUCUGAACAGAUGGAUUACCUU- UUGUCAAAGCAUCAUC UCAACACUGACUUGACUCGACGUCCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGU- ACCCCGAGUCUCCCCC GACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC- CAAGCACGCAGCAAUG CAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAA- GUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAGCAUAUGAC UAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA IL-12 mouse 36 ModA murine MRVTAPRTLILLLSGALALTETWAGSGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHG- VIGSGKTLTITVKEF IL-12 LDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDL- KFNIKSSSSSPDSRAVTCGMA (amino SLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPP- KNLQMKPLKNSQVEVSWEYPDS acid) WSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSK- WACVPCRVRSVPGVGVPGVGR VIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLAT- RETSSTTRGSCLPPQK TSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEAD- PYRVKMKLCILLHAFS TRVVTINRVMGYLSSA 37 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGACCGCCCCCAGA- ACCCTGATCCTGCTG IL-12 CTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGCGGATCCATGTGGGAGCTGGAGAAAG- ACGTTTATGTTGTAGAGGTGG (DNA: ACTGGACTCCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGACAT- CACCTGGACCTCAGACCAGAG 5'UTR- ACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTCTAGATGCTGGCCA- GTACACCTGCCACAAAGGAGGC CDS-3'UTR) GAGACTCTGAGCCACTCACATCTGCTGCTCCACAAGAAGGAAAATGGAATTTGGTCCACTGAAATTTTAAAAA- ATTTCAAAAACAAGA CTTTCCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAGAAACATGG- ACTTGAAGTTCAACAT CAAGAGCAGTAGCAGTTCCCCTGACTCTCGGGCAGTGACATGTGGAATGGCGTCTCTGTCTGCAGAGAAGGT- CACACTGGACCAAAGG GACTATGAGAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCCCATT- GAACTGGCGTTGGAAG CACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTCTTCATCAGGGACATCATCAAACCAGACCCGC- CCAAGAACTTGCAGAT GAAGCCTTTGAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCCATTCCTA- CTTCTCCCTCAAGTTC TTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGGAGACAGAGGAGGGGTGTAACCAGAAAGGTGCGTTC- CTCGTAGAGAAGACAT CTACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTACAATTCCTCATGCA- GCAAGTGGGCATGTGT TCCCTGCAGAGTCCGATCGGTTCCTGGAGTAGGGGTACCTGGAGTGGGCAGGGTCATACCGGTCTCTGGACC- TGCCAGGTGTCTTAGC CAGTCCCGAAACCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAGCTGAAACATTATTCC- TGCACTGCTGAAGACA TCGATCATGAAGACATCACACGGGACCAAACCAGCACATTGAAGACCTGTTTACCACTGGAACTACACAAGA- ACGAGAGTTGCCTGGC TACTAGAGAGACTTCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTTGATGATGACCCT- GTGCCTTGGTAGCATC TATGAGGACTTGAAGATGTACCAGACAGAGTTCCAGGCCATCAACGCAGCACTTCAGAATCACAACCATCAG- CAGATCATTCTAGACA AGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCGAGACTCTGCGCCAGAAAC- CTCCTGTGGGAGAAGC AGACCCTTACAGAGTGAAAATGAAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACCATCAA- CAGGGTGATGGGCTAT CTGTCCAGCGCCTAATAGCTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCC- TAAGTCCAACTACTAA ACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGC- GTCGAGAGCTCGCTTT CTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAA- GGGCCTTGAGCATCTG GATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGAGACCTGGTCCAGAGTCGCTAGCAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAA 38 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGACCGCCCCCAGA- ACCCUGAUCCUGCUG IL-12 (RNA) CUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGCGGAUCCAUGUGGGAGCUGGAGAAAGACGUUU- AUGUUGUAGAGGUGG ACUGGACUCCCGAUGCCCCUGGAGAAACAGUGAACCUCACCUGUGACACGCCUGAAGAAGAUGACAUCACCU- GGACCUCAGACCAGAG ACAUGGAGUCAUAGGCUCUGGAAAGACCCUGACCAUCACUGUCAAAGAGUUUCUAGAUGCUGGCCAGUACAC- CUGCCACAAAGGAGGC GAGACUCUGAGCCACUCACAUCUGCUGCUCCACAAGAAGGAAAAUGGAAUUUGGUCCACUGAAAUUUUAAAA- AAUUUCAAAAACAAGA CUUUCCUGAAGUGUGAAGCACCAAAUUACUCCGGACGGUUCACGUGCUCAUGGCUGGUGCAAAGAAACAUGG- ACUUGAAGUUCAACAU CAAGAGCAGUAGCAGUUCCCCUGACUCUCGGGCAGUGACAUGUGGAAUGGCGUCUCUGUCUGCAGAGAAGGU- CACACUGGACCAAAGG GACUAUGAGAAGUAUUCAGUGUCCUGCCAGGAGGAUGUCACCUGCCCAACUGCCGAGGAGACCCUGCCCAUU- GAACUGGCGUUGGAAG CACGGCAGCAGAAUAAAUAUGAGAACUACAGCACCAGCUUCUUCAUCAGGGACAUCAUCAAACCAGACCCGC- CCAAGAACUUGCAGAU GAAGCCUUUGAAGAACUCACAGGUGGAGGUCAGCUGGGAGUACCCUGACUCCUGGAGCACUCCCCAUUCCUA- CUUCUCCCUCAAGUUC UUUGUUCGAAUCCAGCGCAAGAAAGAAAAGAUGAAGGAGACAGAGGAGGGGUGUAACCAGAAAGGUGCGUUC- CUCGUAGAGAAGACAU CUACCGAAGUCCAAUGCAAAGGCGGGAAUGUCUGCGUGCAAGCUCAGGAUCGCUAUUACAAUUCCUCAUGCA- GCAAGUGGGCAUGUGU UCCCUGCAGAGUCCGAUCGGUUCCUGGAGUAGGGGUACCUGGAGUGGGCAGGGUCAUACCGGUCUCUGGACC- UGCCAGGUGUCUUAGC CAGUCCCGAAACCUGCUGAAGACCACAGAUGACAUGGUGAAGACGGCCAGAGAAAAGCUGAAACAUUAUUCC- UGCACUGCUGAAGACA UCGAUCAUGAAGACAUCACACGGGACCAAACCAGCACAUUGAAGACCUGUUUACCACUGGAACUACACAAGA- ACGAGAGUUGCCUGGC UACUAGAGAGACUUCUUCCACAACAAGAGGGAGCUGCCUGCCCCCACAGAAGACGUCUUUGAUGAUGACCCU- GUGCCUUGGUAGCAUC UAUGAGGACUUGAAGAUGUACCAGACAGAGUUCCAGGCCAUCAACGCAGCACUUCAGAAUCACAACCAUCAG- CAGAUCAUUCUAGACA AGGGCAUGCUGGUGGCCAUCGAUGAGCUGAUGCAGUCUCUGAAUCAUAAUGGCGAGACUCUGCGCCAGAAAC- CUCCUGUGGGAGAAGC AGACCCUUACAGAGUGAAAAUGAAGCUCUGCAUCCUGCUUCACGCCUUCAGCACCCGCGUCGUGACCAUCAA- CAGGGUGAUGGGCUAU CUGUCCAGCGCCUAAUAGCUCGAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCC- UAAGUCCAACUACUAA ACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGC- GUCGAGAGCUCGCUUU CUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAA- GGGCCUUGAGCAUCUG GAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAGACCUGGUCCAGAGUCGCUAGCAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAA 39 ModB murine MGAMAPRTLLLLLAAALAPTQTRAGPGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHG-

VIGSGKTLTITVKEF IL-12 LDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNEKNKTFLKCEAPNYSGRFTCSWLVQRNMDL- KFNIKSSSSSPDSRAVTCGMA (amino SLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPP- KNLQMKPLKNSQVEVSWEYPDS acid) WSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSK- WACVPCRVRSVPGVGVPGVGR VIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLAT- RETSSTTRGSCLPPQK TSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEAD- PYRVKMKLCILLHAFS TRVVTINRVMGYLSSA 40 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA IL-12 TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCA- TGGCCCCTAGAACATTGCTCC (DNA: TGCTGCTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCATGTGGGAGCTGGA- GAAAGACGTTTATGTTGTAGA 5'UTR- GGTGGACTGGACTCCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGA- TGACATCACCTGGACCTCAGAC CDS-3'UTR) CAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTCTAGATGCTGGCCAGT- ACACCTGCCACAAAG GAGGCGAGACTCTGAGCCACTCACATCTGCTGCTCCACAAGAAGGAAAATGGAATTTGGTCCACTGAAATTT- TAAAAAATTTCAAAAA CAAGACTTTCCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAGAAA- CATGGACTTGAAGTTC AACATCAAGAGCAGTAGCAGTTCCCCTGACTCTCGGGCAGTGACATGTGGAATGGCGTCTCTGTCTGCAGAG- AAGGTCACACTGGACC AAAGGGACTATGAGAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGC- CCATTGAACTGGCGTT GGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTCTTCATCAGGGACATCATCAAACCAGA- CCCGCCCAAGAACTTG CAGATGAAGCCTTTGAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCCAT- TCCTACTTCTCCCTCA AGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGGAGACAGAGGAGGGGTGTAACCAGAAAGGTG- CGTTCCTCGTAGAGAA GACATCTACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTACAATTCCTC- ATGCAGCAAGTGGGCA TGTGTTCCCTGCAGAGTCCGATCGGTTCCTGGAGTAGGGGTACCTGGAGTGGGCAGGGTCATACCGGTCTCT- GGACCTGCCAGGTGTC TTAGCCAGTCCCGAAACCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAGCTGAAACATT- ATTCCTGCACTGCTGA AGACATCGATCATGAAGACATCACACGGGACCAAACCAGCACATTGAAGACCTGTTTACCACTGGAACTACA- CAAGAACGAGAGTTGC CTGGCTACTAGAGAGACTTCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTTGATGATG- ACCCTGTGCCTTGGTA GCATCTATGAGGACTTGAAGATGTACCAGACAGAGTTCCAGGCCATCAACGCAGCACTTCAGAATCACAACC- ATCAGCAGATCATTCT AGACAAGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCGAGACTCTGCGCCA- GAAACCTCCTGTGGGA GAAGCAGACCCTTACAGAGTGAAAATGAAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACC- ATCAACAGGGTGATGG GCTATCTGTCCAGCGCCTAATAGCTCGACGTCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCG- TCCTGGGTACCCCGAG TCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAG- ACACCTCCCAAGCACG CAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAAT- AAACGAAAGTTTAACT AAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAG CATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAA 41 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA IL-12 (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCAUGGCCC- CUAGAACAUUGCUCC UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCAUGUGGGAGCUGGAGAAAG- ACGUUUAUGUUGUAGA GGUGGACUGGACUCCCGAUGCCCCUGGAGAAACAGUGAACCUCACCUGUGACACGCCUGAAGAAGAUGACAU- CACCUGGACCUCAGAC CAGAGACAUGGAGUCAUAGGCUCUGGAAAGACCCUGACCAUCACUGUCAAAGAGUUUCUAGAUGCUGGCCAG- UACACCUGCCACAAAG GAGGCGAGACUCUGAGCCACUCACAUCUGCUGCUCCACAAGAAGGAAAAUGGAAUUUGGUCCACUGAAAUUU- UAAAAAAUUUCAAAAA CAAGACUUUCCUGAAGUGUGAAGCACCAAAUUACUCCGGACGGUUCACGUGCUCAUGGCUGGUGCAAAGAAA- CAUGGACUUGAAGUUC AACAUCAAGAGCAGUAGCAGUUCCCCUGACUCUCGGGCAGUGACAUGUGGAAUGGCGUCUCUGUCUGCAGAG- AAGGUCACACUGGACC AAAGGGACUAUGAGAAGUAUUCAGUGUCCUGCCAGGAGGAUGUCACCUGCCCAACUGCCGAGGAGACCCUGC- CCAUUGAACUGGCGUU GGAAGCACGGCAGCAGAAUAAAUAUGAGAACUACAGCACCAGCUUCUUCAUCAGGGACAUCAUCAAACCAGA- CCCGCCCAAGAACUUG CAGAUGAAGCCUUUGAAGAACUCACAGGUGGAGGUCAGCUGGGAGUACCCUGACUCCUGGAGCACUCCCCAU- UCCUACUUCUCCCUCA AGUUCUUUGUUCGAAUCCAGCGCAAGAAAGAAAAGAUGAAGGAGACAGAGGAGGGGUGUAACCAGAAAGGUG- CGUUCCUCGUAGAGAA GACAUCUACCGAAGUCCAAUGCAAAGGCGGGAAUGUCUGCGUGCAAGCUCAGGAUCGCUAUUACAAUUCCUC- AUGCAGCAAGUGGGCA UGUGUUCCCUGCAGAGUCCGAUCGGUUCCUGGAGUAGGGGUACCUGGAGUGGGCAGGGUCAUACCGGUCUCU- GGACCUGCCAGGUGUC UUAGCCAGUCCCGAAACCUGCUGAAGACCACAGAUGACAUGGUGAAGACGGCCAGAGAAAAGCUGAAACAUU- AUUCCUGCACUGCUGA AGACAUCGAUCAUGAAGACAUCACACGGGACCAAACCAGCACAUUGAAGACCUGUUUACCACUGGAACUACA- CAAGAACGAGAGUUGC CUGGCUACUAGAGAGACUUCUUCCACAACAAGAGGGAGCUGCCUGCCCCCACAGAAGACGUCUUUGAUGAUG- ACCCUGUGCCUUGGUA GCAUCUAUGAGGACUUGAAGAUGUACCAGACAGAGUUCCAGGCCAUCAACGCAGCACUUCAGAAUCACAACC- AUCAGCAGAUCAUUCU AGACAAGGGCAUGCUGGUGGCCAUCGAUGAGCUGAUGCAGUCUCUGAAUCAUAAUGGCGAGACUCUGCGCCA- GAAACCUCCUGUGGGA GAAGCAGACCCUUACAGAGUGAAAAUGAAGCUCUGCAUCCUGCUUCACGCCUUCAGCACCCGCGUCGUGACC- AUCAACAGGGUGAUGG GCUAUCUGUCCAGCGCCUAAUAGCUCGACGUCCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG- UCCUGGGUACCCCGAG UCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAG- ACACCUCCCAAGCACG CAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAU- AAACGAAAGUUUAACU AAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAG CAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAA IFN.alpha. (IFN.alpha.4) mouse 42 ModA murine MRVTAPRTLILLLSGALALTETWAGSGSCDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDN- QQIQKAQAILVLRDL IFN.alpha.4 TQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSL- CAWEVIRAEVWRALS (amino SSTNLLARLSEEKE acid) 43 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGACCGCCCCCAGA- ACCCTGATCCTGCTG IFN.alpha.4 CTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGCGGATCCTGTGACCTGCCTCACACTTATAACC- TCGGGAACAAGAGGG (DNA: CCTTGACAGTCCTGGAAGAAATGAGAAGACTCCCCCCTCTTTCCTGCCTGAAGGACAGGAAGGATTT- TGGATTCCCCTTGGAGAAGGT 5'UTR- GGATAACCAACAGATCCAGAAGGCTCAAGCCATCCTTGTGCTAAGAGATCTTACCCAGCAGATTTT- GAACCTCTTCACATCAAAAGAC CDS-3'UTR) TTGTCTGCTACTTGGAATGCAACTCTCCTAGACTCATTCTGCAATGACCTCCATCAGCAGCTCAATGATCTCA- AAGCCTGTGTGATGC AGGAACCTCCTCTGACCCAGGAAGACTCCCTGCTGGCTGTGAGGACATACTTCCACAGGATCACTGTGTACC- TGAGAAAGAAGAAACA CAGCCTCTGTGCCTGGGAGGTGATCAGAGCAGAAGTCTGGAGAGCCCTCTCTTCCTCAACCAACTTGCTGGC- AAGACTGAGTGAGGAG AAGGAGTGATAACTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTC- CAACTACTAAACTGGG GGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGAG- AGCTCGCTTTCTTGCT GTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCT- TGAGCATCTGGATTCT GCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGAGACCTGGTCCAGAGTCGCTAGCAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAA 44 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGACCGCCCCCAGA- ACCCUGAUCCUGCUG IFN.alpha.4 (RNA) CUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGCGGAUCCUGUGACCUGCCUCACACUUAUAACC- UCGGGAACAAGAGGG CCUUGACAGUCCUGGAAGAAAUGAGAAGACUCCCCCCUCUUUCCUGCCUGAAGGACAGGAAGGAUUUUGGAU- UCCCCUUGGAGAAGGU GGAUAACCAACAGAUCCAGAAGGCUCAAGCCAUCCUUGUGCUAAGAGAUCUUACCCAGCAGAUUUUGAACCU- CUUCACAUCAAAAGAC UUGUCUGCUACUUGGAAUGCAACUCUCCUAGACUCAUUCUGCAAUGACCUCCAUCAGCAGCUCAAUGAUCUC- AAAGCCUGUGUGAUGC AGGAACCUCCUCUGACCCAGGAAGACUCCCUGCUGGCUGUGAGGACAUACUUCCACAGGAUCACUGUGUACC- UGAGAAAGAAGAAACA CAGCCUCUGUGCCUGGGAGGUGAUCAGAGCAGAAGUCUGGAGAGCCCUCUCUUCCUCAACCAACUUGCUGGC- AAGACUGAGUGAGGAG AAGGAGUGAUAACUCGAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUC- CAACUACUAAACUGGG GGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAG- AGCUCGCUUUCUUGCU GUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCU- UGAGCAUCUGGAUUCU GCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAGACCUGGUCCAGAGUCGCUAGCAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAA 45 ModB murine MGAMAPRTLLLLLAAALAPTQTRAGPGSCDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDN- QQIQKAQAILVLRDL IFN.alpha.4 TQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSL- CAWEVIRAEVWRALS (amino SSTNLLARLSEEKE acid) 46 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA IFN.alpha.4 TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCATGGCCC- CTAGAACATTGCTCC (DNA: TGCTGCTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCTGTGACCTGCCTCA- CACTTATAACCTCGGGAACAA 5'UTR- GAGGGCCTTGACAGTCCTGGAAGAAATGAGAAGACTCCCCCCTCTTTCCTGCCTGAAGGACAGGAA- GGATTTTGGATTCCCCTTGGAG CDS-3'UTR) AAGGTGGATAACCAACAGATCCAGAAGGCTCAAGCCATCCTTGTGCTAAGAGATCTTACCCAGCAGATTTTGA- ACCTCTTCACATCAA AAGACTTGTCTGCTACTTGGAATGCAACTCTCCTAGACTCATTCTGCAATGACCTCCATCAGCAGCTCAATG- ATCTCAAAGCCTGTGT GATGCAGGAACCTCCTCTGACCCAGGAAGACTCCCTGCTGGCTGTGAGGACATACTTCCACAGGATCACTGT- GTACCTGAGAAAGAAG AAACACAGCCTCTGTGCCTGGGAGGTGATCAGAGCAGAAGTCTGGAGAGCCCTCTCTTCCTCAACCAACTTG- CTGGCAAGACTGAGTG AGGAGAAGGAGTGATAACTCGACGTCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGG- GTACCCCGAGTCTCCC CCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCT- CCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGA- AAGTTTAACTAAGCTA TACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAGCATATG ACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- A 47 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA IFN.alpha.4 (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCAUGGCCC- CUAGAACAUUGCUCC UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCUGUGACCUGCCUCACACUU- AUAACCUCGGGAACAA GAGGGCCUUGACAGUCCUGGAAGAAAUGAGAAGACUCCCCCCUCUUUCCUGCCUGAAGGACAGGAAGGAUUU- UGGAUUCCCCUUGGAG AAGGUGGAUAACCAACAGAUCCAGAAGGCUCAAGCCAUCCUUGUGCUAAGAGAUCUUACCCAGCAGAUUUUG- AACCUCUUCACAUCAA AAGACUUGUCUGCUACUUGGAAUGCAACUCUCCUAGACUCAUUCUGCAAUGACCUCCAUCAGCAGCUCAAUG- AUCUCAAAGCCUGUGU GAUGCAGGAACCUCCUCUGACCCAGGAAGACUCCCUGCUGGCUGUGAGGACAUACUUCCACAGGAUCACUGU- GUACCUGAGAAAGAAG AAACACAGCCUCUGUGCCUGGGAGGUGAUCAGAGCAGAAGUCUGGAGAGCCCUCUCUUCCUCAACCAACUUG- CUGGCAAGACUGAGUG AGGAGAAGGAGUGAUAACUCGACGUCCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGG- GUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCU- CCCAAGCACGCAGCAA UGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGA- AAGUUUAACUAAGCUA UACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAGCAUAUG ACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- A

IL-15 sushi mouse 48 ModA murine MGAMAPRTLLLLLAAALAPTQTRAGPGSTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIEC- VINKNTNVAHWTTPS IL-15 sushi LKCIRDPSLAGGSGGSGGSGGSGGSGGSGGNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCF- LLELQVILHEYSNMT (amino LNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS acid) 49 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGCGCCATGGCCCCTAGA- ACATTGCTCCTGCTG IL-15 sushi CTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCACCACGTGTCCACCTCCCGTATCTA- TTGAGCATGCTGACA (DNA: TCCGGGTCAAGAATTACAGTGTGAACTCCAGGGAGAGGTATGTCTGTAACTCTGGCTTTAAGCGGAA- AGCTGGAACATCCACCCTGAT 5'UTR- TGAGTGTGTGATCAACAAGAACACAAATGTTGCCCACTGGACAACTCCCAGCCTCAAGTGCATCAG- AGACCCCTCCCTAGCTGGAGGG CDS-3'UTR) AGCGGAGGCTCTGGCGGAAGCGGCGGGTCTGGAGGCTCCGGGGGAAGCGGCGGAAATTGGATCGACGTGCGCT- ACGACCTGGAAAAGA TCGAGAGCCTGATCCAGAGCATCCACATCGACACCACCCTGTACACCGACAGCGACTTCCACCCCAGCTGCA- AAGTGACCGCTATGAA CTGCTTCCTGCTGGAACTGCAAGTGATCCTGCACGAGTACAGCAACATGACCCTGAACGAGACAGTGCGGAA- CGTGCTGTACCTGGCC AACAGCACCCTGAGCAGCAACAAGAACGTGGCCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAAAAG- ACCTTCACCGAGTTTC TGCAGAGCTTCATCAGGATCGTGCAGATGTTCATCAACACCTCTTGATGAGTCGACGTCCTGGTACTGCATG- CACGCAATGCTAGCTG CCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGC- CCCACTCACCACCTCT GCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGG- AAACAGCAGTGATTAA CCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACAC- CCTCGAGCTAGCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA 50 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGCCAUGGCCCCUAGA- ACAUUGCUCCUGCUG IL-15 sushi CUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCACCACGUGUCCACCUCCCGUAUCUA- UUGAGCAUGCUGACA (RNA) UCCGGGUCAAGAAUUACAGUGUGAACUCCAGGGAGAGGUAUGUCUGUAACUCUGGCUUUAAGCGGAA- AGCUGGAACAUCCACCCUGAU UGAGUGUGUGAUCAACAAGAACACAAAUGUUGCCCACUGGACAACUCCCAGCCUCAAGUGCAUCAGAGACCC- CUCCCUAGCUGGAGGG AGCGGAGGCUCUGGCGGAAGCGGCGGGUCUGGAGGCUCCGGGGGAAGCGGCGGAAAUUGGAUCGACGUGCGC- UACGACCUGGAAAAGA UCGAGAGCCUGAUCCAGAGCAUCCACAUCGACACCACCCUGUACACCGACAGCGACUUCCACCCCAGCUGCA- AAGUGACCGCUAUGAA CUGCUUCCUGCUGGAACUGCAAGUGAUCCUGCACGAGUACAGCAACAUGACCCUGAACGAGACAGUGCGGAA- CGUGCUGUACCUGGCC AACAGCACCCUGAGCAGCAACAAGAACGUGGCCGAGAGCGGCUGCAAAGAGUGCGAGGAACUGGAAGAAAAG- ACCUUCACCGAGUUUC UGCAGAGCUUCAUCAGGAUCGUGCAGAUGUUCAUCAACACCUCUUGAUGAGUCGACGUCCUGGUACUGCAUG- CACGCAAUGCUAGCUG CCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC- CCCACUCACCACCUCU GCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG- AAACAGCAGUGAUUAA CCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACAC- CCUCGAGCUAGCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA 51 ModB murine MGAMAPRTLLLLLAAALAPTQTRAGPGSTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIEC- VINKNTNVAHWTTPS IL-15 sushi LKCIRDPSLAGGSGGSGGSGGSGGSGGSGGNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCF- LLELQVILHEYSNMT (amino LNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS acid) 52 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA IL-15 sushi TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCATGGCCC- CTAGAACATTGCTCC (DNA: TGCTGCTGGCCGCTGCCCTGGCCCCTACZCAGACAAGAGCTGGACCTGGATCCACCACGTGTCCACC- TCCCGTATCTATTGAGCATGC 5'UTR- TGACATCCGGGTCAAGAATTACAGTGTGAACTCCAGGGAGAGGTATGTCTGTAACTCTGGCTTTAA- GCGGAAAGCTGGAACATCCACC CDS-3'UTR) CTGATTGAGTGTGTGATCAACAAGAACACAAATGTTGCCCACTGGACAACTCCCAGCCTCAAGTGCATCAGAG- ACCCCTCCCTAGCTG GAGGGAGCGGAGGCTCTGGCGGAAGCGGCGGGTCTGGAGGCTCCGGGGGAAGCGGCGGAAATTGGATCGACG- TGCGCTACGACCTGGA AAAGATCGAGAGCCTGATCCAGAGCATCCACATCGACACCACCCTGTACACCGACAGCGACTTCCACCCCAG- CTGCAAAGTGACCGCT ATGAACTGCTTCCTGCTGGAACTGCAAGTGATCCTGCACGAGTACAGCAACATGACCCTGAACGAGACAGTG- CGGAACGTGCTGTACC TGGCCAACAGCACCCTGAGCAGCAACAAGAACGTGGCCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAG- AAAAGACCTTCACCGA GTTTCTGCAGAGCTTCATCAGGATCGTGCAGATGTTCATCAACACCTCTTGATGAGTCGACGTCCTGGTACT- GCATGCACGCAATGCT AGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCA- CCTGCCCCACTCACCA CCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCC- ACGGGAAACAGCAGTG ATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGC- CACACCCTCGAGCTAG CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA 53 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA IL-15 sushi UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCAUGGCCC- CUAGAACAUUGCUCC (RNA) UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCACCACGUGUCCACC- UCCCGUAUCUAUUGAGCAUGC UGACAUCCGGGUCAAGAAUUACAGUGUGAACUCCAGGGAGAGGUAUGUCUGUAACUCUGGCUUUAAGCGGAA- AGCUGGAACAUCCACC CUGAUUGAGUGUGUGAUCAACAAGAACACAAAUGUUGCCCACUGGACAACUCCCAGCCUCAAGUGCAUCAGA- GACCCCUCCCUAGCUG GAGGGAGCGGAGGCUCUGGCGGAAGCGGCGGGUCUGGAGGCUCCGGGGGAAGCGGCGGAAAUUGGAUCGACG- UGCGCUACGACCUGGA AAAGAUCGAGAGCCUGAUCCAGAGCAUCCACAUCGACACCACCCUGUACACCGACAGCGACUUCCACCCCAG- CUGCAAAGUGACCGCU AUGAACUGCUUCCUGCUGGAACUGCAAGUGAUCCUGCACGAGUACAGCAACAUGACCCUGAACGAGACAGUG- CGGAACGUGCUGUACC UGGCCAACAGCACCCUGAGCAGCAACAAGAACGUGGCCGAGAGCGGCUGCAAAGAGUGCGAGGAACUGGAAG- AAAAGACCUUCACCGA GUUUCUGCAGAGCUUCAUCAGGAUCGUGCAGAUGUUCAUCAACACCUCUUGAUGAGUCGACGUCCUGGUACU- GCAUGCACGCAAUGCU AGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCA- CCUGCCCCACUCACCA CCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCC- ACGGGAAACAGCAGUG AUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGC- CACACCCUCGAGCUAG CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA GM-CSF mouse 54 ModA murine MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRL- KIFEQGLRGNFTKLK GM-CSF GALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK (amino acid) 55 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTGGCTGCAGAACCTGCTG- TTCCTGGGCATCGTG GM-CSF GTGTACAGCCTGAGCGCCCCCACCAGGAGCCCCATCACCGTGACCAGGCCCTGGAAGCACGTGGAG- GCCATCAAGGAGGCCCTGAACC (DNA: TGCTGGACGACATGCCCGTGACCCTGAACGAGGAGGTGGAGGTGGTGAGCAACGAGTTCAGCTTCAA- GAAGCTGACCTGCGTGCAGAC 5'UTR- CAGGCTGAAGATCTTCGAGCAGGGCCTGAGGGGCAACTTCACCAAGCTGAAGGGCGCCCTGAACAT- GACCGCCAGCTACTACCAGACC CDS-3'UTR) TACTGCCCCCCCACCCCCGAGACCGACTGCGAGACCCAGGTGACCACCTACGCCGACTTCATCGACAGCCTGA- AGACCTTCCTGACCG ACATCCCCTTCGAGTGCAAGAAGCCCGGCCAGAAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTA- GCTGCCCCTTTCCCGT CCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCAC- CTCTGCTAGTTCCAGA CACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGA- TTAACCTTTAGCAATA AACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTC- CAGAGTCGCTAGCCGC GTCGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA 56 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGUGGCUGCAGAACCUGCUG- UUCCUGGGCAUCGUG GM-CSF GUGUACAGCCUGAGCGCCCCCACCAGGAGCCCCAUCACCGUGACCAGGCCCUGGAAGCACGUGGAG- GCCAUCAAGGAGGCCCUGAACC (RNA) UGCUGGACGACAUGCCCGUGACCCUGAACGAGGAGGUGGAGGUGGUGAGCAACGAGUUCAGCUUCAA- GAAGCUGACCUGCGUGCAGAC CAGGCUGAAGAUCUUCGAGCAGGGCCUGAGGGGCAACUUCACCAAGCUGAAGGGCGCCCUGAACAUGACCGC- CAGCUACUACCAGACC UACUGCCCCCCCACCCCCGAGACCGACUGCGAGACCCAGGUGACCACCUACGCCGACUUCAUCGACAGCCUG- AAGACCUUCCUGACCG ACAUCCCCUUCGAGUGCAAGAAGCCCGGCCAGAAGUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUA- GCUGCCCCUUUCCCGU CCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCAC- CUCUGCUAGUUCCAGA CACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGA- UUAACCUUUAGCAAUA AACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUC- CAGAGUCGCUAGCCGC GUCGCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA 57 ModB murine MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRL- KIFEQGLRGNFTKLK GM-CSF GALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK (amino acid) 58 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA GM-CSF TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGTGGCTG- CAGAACCTGCTGTTCCTGGGCA (DNA: TCGTGGTGTACAGCCTGAGCGCCCCCACCAGGAGCCCCATCACCGTGACCAGGCCCTGGAAGCACGT- GGAGGCCATCAAGGAGGCCCT 5'UTR- GAACCTGCTGGACGACATGCCCGTGACCCTGAACGAGGAGGTGGAGGTGGTGAGCAACGAGTTCAG- CTTCAAGAAGCTGACCTGCGTG CDS-3'UTR) CAGACCAGGCTGAAGATCTTCGAGCAGGGCCTGAGGGGCAACTTCACCAAGCTGAAGGGCGCCCTGAACATGA- CCGCCAGCTACTACC AGACCTACTGCCCCCCCACCCCCGAGACCGACTGCGAGACCCAGGTGACCACCTACGCCGACTTCATCGACA- GCCTGAAGACCTTCCT GACCGACATCCCCTTCGAGTGCAAGAAGCCCGGCCAGAAGTGATGACTCGAGCTGGTACTGCATGCACGCAA- TGCTAGCTGCCCCTTT CCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTC- ACCACCTCTGCTAGTT CCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGC- AGTGATTAACCTTTAG CAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACC- TGGTCCAGAGTCGCTA GCCGCGTCGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 59 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA GM-CSF UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGUGGCUG- CAGAACCUGCUGUUCCUGGGCA (RNA) UCGUGGUGUACAGCCUGAGCGCCCCCACCAGGAGCCCCAUCACCGUGACCAGGCCCUGGAAGCACGU- GGAGGCCAUCAAGGAGGCCCU GAACCUGCUGGACGACAUGCCCGUGACCCUGAACGAGGAGGUGGAGGUGGUGAGCAACGAGUUCAGCUUCAA- GAAGCUGACCUGCGUG CAGACCAGGCUGAAGAUCUUCGAGCAGGGCCUGAGGGGCAACUUCACCAAGCUGAAGGGCGCCCUGAACAUG- ACCGCCAGCUACUACC AGACCUACUGCCCCCCCACCCCCGAGACCGACUGCGAGACCCAGGUGACCACCUACGCCGACUUCAUCGACA- GCCUGAAGACCUUCCU GACCGACAUCCCCUUCGAGUGCAAGAAGCCCGGCCAGAAGUGAUGACUCGAGCUGGUACUGCAUGCACGCAA- UGCUAGCUGCCCCUUU CCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUC- ACCACCUCUGCUAGUU

CCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGC- AGUGAUUAACCUUUAG CAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACC- UGGUCCAGAGUCGCUA GCCGCGUCGCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FLT3L mouse 60 ModA FLT3L MGAMAPRTLLLLLAAALAPTQTRAGPGSTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCG- GLWRLVLAQRWMERL (amino KTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSR- CLELQCQPDSSTLPPPWSPRPL acid, human EATAPTAPQPP FLT3L in combination with a mouse optimized secretion sequence)) 61 ModA FLT3L GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGCGCCATGGCCCCTAGA- ACATTGCTCCTGCTG (DNA: CTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCACCCAGGACTGCAGCTTCC- AGCACTCCCCTATCTCCTCCG 5'UTR- ACTTCGCCGTGAAGATCCGGGAGCTGTCCGATTACCTGCTGCAGGACTACCCTGTGACCGTGGCCA- GCAACCTGCAGGACGAAGAACT CDS-3'UTR) GTGTGGCGGCCTGTGGCGGCTGGTGCTGGCCCAGCGGTGGATGGAACGGCTGAAAACCGTGGCCGGCTCCAAG- ATGCAGGGCCTGCTC GAGCGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCTTCCTGCCTGCGGTTC- GTGCAGACCAACATCT CCCGGCTGCTGCAGGAAACCTCCGAGCAGCTGGTCGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCCC- GGTGTCTGGAACTCCA GTGTCAGCCCGACTCCTCCACCCTGCCTCCTCCCTGGTCCCCCAGGCCTCTGGAAGCCACCGCCCCTACCGC- CCCACAGCCTCCTTGA TAGGTCGACGTCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTC- CCCCGACCTCGGGTCC CAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGC- AATGCAGCTCAAAACG CTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGC- TATACTAACCCCAGGG TTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATA- TGACTAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 62 ModA FLT3L GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGCCAUGGCCCCUAGA- ACAUUGCUCCUGCUG (RNA) CUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCACCCAGGACUGCAGCUUCC- AGCACUCCCCUAUCUCCUCCG ACUUCGCCGUGAAGAUCCGGGAGCUGUCCGAUUACCUGCUGCAGGACUACCCUGUGACCGUGGCCAGCAACC- UGCAGGACGAAGAACU GUGUGGCGGCCUGUGGCGGCUGGUGCUGGCCCAGCGGUGGAUGGAACGGCUGAAAACCGUGGCCGGCUCCAA- GAUGCAGGGCCUGCUC GAGCGGGUGAACACCGAGAUCCACUUCGUGACCAAGUGCGCCUUCCAGCCUCCUCCUUCCUGCCUGCGGUUC- GUGCAGACCAACAUCU CCCGGCUGCUGCAGGAAACCUCCGAGCAGCUGGUCGCCCUGAAGCCUUGGAUCACCCGGCAGAACUUCUCCC- GGUGUCUGGAACUCCA GUGUCAGCCCGACUCCUCCACCCUGCCUCCUCCCUGGUCCCCCAGGCCUCUGGAAGCCACCGCCCCUACCGC- CCCACAGCCUCCUUGA UAGGUCGACGUCCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUC- CCCCGACCUCGGGUCC CAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGC- AAUGCAGCUCAAAACG CUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC- UAUACUAACCCCAGGG UUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUA- UGACUAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 63 ModB FLT3L MGAMAPRTLLLLLAAALAPTQTRAGPGSTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCG- GLWRLVLAQRWMERL (amino KTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSR- CLELQCQPDSSTLPPPWSPRPL acid, human EATAPTAPQPP FLT3L in combination with a mouse optimized secretion sequence) 64 ModB FLT3L GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA (DNA: TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCA- TGGCCCCTAGAACATTGCTCC 5'UTR- TGCTGCTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCACCCAGGACTGCA- GCTTCCAGCACTCCCCTATCTC CDS-3'UTR) CTCCGACTTCGCCGTGAAGATCCGGGAGCTGTCCGATTACCTGCTGCAGGACTACCCTGTGACCGTGGCCAGC- AACCTGCAGGACGAA GAACTGTGTGGCGGCCTGTGGCGGCTGGTGCTGGCCCAGCGGTGGATGGAACGGCTGAAAACCGTGGCCGGC- TCCAAGATGCAGGGCC TGCTCGAGCGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCTTCCTGCCTGC- GGTTCGTGCAGACCAA CATCTCCCGGCTGCTGCAGGAAACCTCCGAGCAGCTGGTCGCCCTGAAGCCTTGGATCACCCGGCAGAACTT- CTCCCGGTGTCTGGAA CTCCAGTGTCAGCCCGACTCCTCCACCCTGCCTCCTCCCTGGTCCCCCAGGCCTCTGGAAGCCACCGCCCCT- ACCGCCCCACAGCCTC CTTGATAGGTCGACGTCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA- GTCTCCCCCGACCTCG GGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCAC- GCAGCAATGCAGCTCA AAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAAC- TAAGCTATACTAACCC CAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- GCATATGACTAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 65 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA FLT3L (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCAUGGCCC- CUAGAACAUUGCUCC UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCACCCAGGACUGCAGCUUCC- AGCACUCCCCUAUCUC CUCCGACUUCGCCGUGAAGAUCCGGGAGCUGUCCGAUUACCUGCUGCAGGACUACCCUGUGACCGUGGCCAG- CAACCUGCAGGACGAA GAACUGUGUGGCGGCCUGUGGCGGCUGGUGCUGGCCCAGCGGUGGAUGGAACGGCUGAAAACCGUGGCCGGC- UCCAAGAUGCAGGGCC UGCUCGAGCGGGUGAACACCGAGAUCCACUUCGUGACCAAGUGCGCCUUCCAGCCUCCUCCUUCCUGCCUGC- GGUUCGUGCAGACCAA CAUCUCCCGGCUGCUGCAGGAAACCUCCGAGCAGCUGGUCGCCCUGAAGCCUUGGAUCACCCGGCAGAACUU- CUCCCGGUGUCUGGAA CUCCAGUGUCAGCCCGACUCCUCCACCCUGCCUCCUCCCUGGUCCCCCAGGCCUCUGGAAGCCACCGCCCCU- ACCGCCCCACAGCCUC CUUGAUAGGUCGACGUCCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGA- GUCUCCCCCGACCUCG GGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCAC- GCAGCAAUGCAGCUCA AAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAAC- UAAGCUAUACUAACCC CAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- GCAUAUGACUAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 41BBL mouse 66 ModA murine MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALLSDTVRPTNAALPTDAAYPAVNVRDREAAWPPA- LNFCSRHPKLYGLVA 41BBL LVLLLLIAACVPIFTRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKN- QASLCNTTLNWHSQDGAGSSY (amino LSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVE- LFPCSMENKLVDRSWSQLLLLK acid) AGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE 67 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGACCAGCACACACTTGAT- GTGGAGGATACCGCG 41BBL GATGCCAGACATCCAGCAGGTACTTCGTGCCCCTCGGATGCGGCGCTCCTCAGAGATACCGGGCTCC- TCGCGGACGCTGCGCTCCTCT (DNA: CAGATACTGTGCGCCCCACAAATGCCGCGCTCCCCACGGATGCTGCCTACCCTGCGGTTAATGTTCG- GGATCGCGAGGCCGCGTGGCC 5'UTR- GCCTGCACTGAACTTCTGTTCCCGCCACCCAAAGCTCTATGGCCTAGTCGCTTTGGTTTTGCTGCT- TCTGATCGCCGCCTGTGTTCCT CDS-3'UTR) ATCTTCACCCGCACCGAGCCTCGGCCAGCGCTCACAATCACCACCTCGCCCAACCTGGGTACCCGAGAGAATA- ATGCAGACCAGGTCA CCCCTGTTTCCCACATTGGCTGCCCCAACACTACACAACAGGGCTCTCCTGTGTTCGCCAAGCTACTGGCTA- AAAACCAAGCATCGTT GTGCAATACAACTCTGAACTGGCACAGCCAAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCTGAGGTA- CGAAGAAGACAAAAAG GAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTTGGAACTGAAGCTCAGTCCAACATTCACAAAC- ACAGGCCACAAGGTGC AGGGCTGGGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTGACAACTTGGCCCTGACAGTGG- AACTGTTCCCTTGCTC CATGGAGAACAAGTTAGTGGACCGTTCCTGGAGTCAACTGTTGCTCCTGAAGGCTGGCCACCGCCTCAGTGT- GGGTCTGAGGGCTTAT CTGCATGGAGCCCAGGATGCATACAGAGACTGGGAGCTGTCTTATCCCAACACCACCAGCTTTGGACTCTTT- CTTGTGAAACCCGACA ACCCATGGGAATGATAGGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGG- GTACCCCGAGTCTCCC CCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCT- CCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGA- AAGTTTAACTAAGCTA TACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAGCATATG ACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- A 68 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGACCAGCACACACUUGAU- GUGGAGGAUACCGCG 41BBL (RNA) GAUGCCAGACAUCCAGCAGGUACUUCGUGCCCCUCGGAUGCGGCGCUCCUCAGAGAUACCGGGCUCCUCGCGG- ACGCUGCGCUCCUCU CAGAUACUGUGCGCCCCACAAAUGCCGCGCUCCCCACGGAUGCUGCCUACCCUGCGGUUAAUGUUCGGGAUC- GCGAGGCCGCGUGGCC GCCUGCACUGAACUUCUGUUCCCGCCACCCAAAGCUCUAUGGCCUAGUCGCUUUGGUUUUGCUGCUUCUGAU- CGCCGCCUGUGUUCCU AUCUUCACCCGCACCGAGCCUCGGCCAGCGCUCACAAUCACCACCUCGCCCAACCUGGGUACCCGAGAGAAU- AAUGCAGACCAGGUCA CCCCUGUUUCCCACAUUGGCUGCCCCAACACUACACAACAGGGCUCUCCUGUGUUCGCCAAGCUACUGGCUA- AAAACCAAGCAUCGUU GUGCAAUACAACUCUGAACUGGCACAGCCAAGAUGGAGCUGGGAGCUCAUACCUAUCUCAAGGUCUGAGGUA- CGAAGAAGACAAAAAG GAGUUGGUGGUAGACAGUCCCGGGCUCUACUACGUAUUUUUGGAACUGAAGCUCAGUCCAACAUUCACAAAC- ACAGGCCACAAGGUGC AGGGCUGGGUCUCUCUUGUUUUGCAAGCAAAGCCUCAGGUAGAUGACUUUGACAACUUGGCCCUGACAGUGG- AACUGUUCCCUUGCUC CAUGGAGAACAAGUUAGUGGACCGUUCCUGGAGUCAACUGUUGCUCCUGAAGGCUGGCCACCGCCUCAGUGU- GGGUCUGAGGGCUUAU CUGCAUGGAGCCCAGGAUGCAUACAGAGACUGGGAGCUGUCUUAUCCCAACACCACCAGCUUUGGACUCUUU- CUUGUGAAACCCGACA ACCCAUGGGAAUGAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGG- GUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCU- CCCAAGCACGCAGCAA UGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGA- AAGUUUAACUAAGCUA UACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAGCAUAUG ACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- A 69 ModB murine MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALLSDTVRPTNAALPTDAAYPAVNVRDREAAWPPA- LNFCSRHPKLYGLVA 41BBL LVLLLLIAACVPIFTRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKN- QASLCNTTLNWHSQDGAGSSY (amino LSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVE- LFPCSMENKLVDRSWSQLLLLK acid) AGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE 70 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA 41BBL TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGACCAGC- ACACACTTGATGTGGAGGATA (DNA: CCGCGGATGCCAGACATCCAGCAGGTACTTCGTGCCCCTCGGATGCGGCGCTCCTCAGAGATACCGG- GCTCCTCGCGGACGCTGCGCT 5'UTR- CCTCTCAGATACTGTGCGCCCCACAAATGCCGCGCTCCCCACGGATGCTGCCTACCCTGCGGTTAA- TGTTCGGGATCGCGAGGCCGCG CDS-3'UTR) TGGCCGCCTGCACTGAACTTCTGTTCCCGCCACCCAAAGCTCTATGGCCTAGTCGCTTTGGTTTTGCTGCTTC- TGATCGCCGCCTGTG TTCCTATCTTCACCCGCACCGAGCCTCGGCCAGCGCTCACAATCACCACCTCGCCCAACCTGGGTACCCGAG- AGAATAATGCAGACCA GGTCACCCCTGTTTCCCACATTGGCTGCCCCAACACTACACAACAGGGCTCTCCTGTGTTCGCCAAGCTACT- GGCTAAAAACCAAGCA TCGTTGTGCAATACAACTCTGAACTGGCACAGCCAAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCTG- AGGTACGAAGAAGACA

AAAAGGAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTTGGAACTGAAGCTCAGTCCAACATTCA- CAAACACAGGCCACAA GGTGCAGGGCTGGGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTGACAACTTGGCCCTGAC- AGTGGAACTGTTCCCT TGCTCCATGGAGAACAAGTTAGTGGACCGTTCCTGGAGTCAACTGTTGCTCCTGAAGGCTGGCCACCGCCTC- AGTGTGGGTCTGAGGG CTTATCTGCATGGAGCCCAGGATGCATACAGAGACTGGGAGCTGTCTTATCCCAACACCACCAGCTTTGGAC- TCTTTCTTGTGAAACC CGACAACCCATGGGAATGATAGGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGT- CCTGGGTACCCCGAGT CTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGA- CACCTCCCAAGCACGC AGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATA- AACGAAAGTTTAACTA AGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAGC ATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAA 71 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUDG- CAGCAAUUUAAAUCA 41BBL (RNA) UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGACCAGCACACAC- UUGAUGUGGAGGAUA CCGCGGAUGCCAGACAUCCAGCAGGUACUUCGUGCCCCUCGGAUGCGGCGCUCCUCAGAGAUACCGGGCUCC- UCGCGGACGCUGCGCU CCUCUCAGAUACUGUGCGCCCCACAAAUGCCGCGCUCCCCACGGAUGCUGCCUACCCUGCGGUUAAUGUUCG- GGAUCGCGAGGCCGCG UGGCCGCCUGCACUGAACUUCUGUUCCCGCCACCCAAAGCUCUAUGGCCUAGUCGCUUUGGUUUUGCUGCUU- CUGAUCGCCGCCUGUG UUCCUAUCUUCACCCGCACCGAGCCUCGGCCAGCGCUCACAAUCACCACCUCGCCCAACCUGGGUACCCGAG- AGAAUAAUGCAGACCA GGUCACCCCUGUUUCCCACAUUGGCUGCCCCAACACUACACAACAGGGCUCUCCUGUGUUCGCCAAGCUACU- GGCUAAAAACCAAGCA UCGUUGUGCAAUACAACUCUGAACUGGCACAGCCAAGAUGGAGCUGGGAGCUCAUACCUAUCUCAAGGUCUG- AGGUACGAAGAAGACA AAAAGGAGUUGGUGGUAGACAGUCCCGGGCUCUACUACGUAUUUUUGGAACUGAAGCUCAGUCCAACAUUCA- CAAACACAGGCCACAA GGUGCAGGGCUGGGUCUCUCUUGUUUUGCAAGCAAAGCCUCAGGUAGAUGACUUUGACAACUUGGCCCUGAC- AGUGGAACUGUUCCCU UGCUCCAUGGAGAACAAGUUAGUGGACCGUUCCUGGAGUCAACUGUUGCUCCUGAAGGCUGGCCACCGCCUC- AGUGUGGGUCUGAGGG CUUAUCUGCAUGGAGCCCAGGAUGCAUACAGAGACUGGGAGCUGUCUUAUCCCAACACCACCAGCUUUGGAC- UCUUUCUUGUGAAACC CGACAACCCAUGGGAAUGAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGU- CCUGGGUACCCCGAGU CUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGA- CACCUCCCAAGCACGC AGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUA- AACGAAAGUUUAACUA AGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAGC AUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAA CD27L-CD40L mouse 72 ModA murine MRVTAPRTLILLLSGALALTETWAGSGSHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHL- RIHQDGLYRLHIQVT CD27L-CD40L LANCSSPGSTLQHRATLAVGICSPAAHGISLLRGRFGQDCTVALQRLTYLVHGDVLCTNLTLPLLPSRNADET- FFGVQWICPGGGSGG (amino GHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCS- SPGSTLQHRATLAVGICSPAAH acid) GISLLRGRFGQDCTVALQRLTYLVHGDVLCTNLTLPLLPSRNADETFFGVQWICPGGGSGGGHPEPH- TAELQLNLTVPRKDPTLRWGA Sequence GPALGRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCSSPGSTLQHRATLAVGICSPAAHGIS- LLRGRFGQDCTVALQRLTYLVHGD annotations VLCTNLTLPLLPSRNADETFEGVQWICPGGGGSGGGGSGGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGY- YTMKSNLVMLENGKQ CAPS: LTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGG- VFELQAGASVFVNVTEASQVI CD27L; HRVGFSSFGLLKLGGGSGGGGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQL- TVKREGLYYVYTQVTFCSNREP CAPS: SSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHR- VGFSSFGLLKLGGGSGGGGDE linker; DPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREP- SSQRPFIVGLWLKPSSGSERILL CAPS: CD40L KAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL 73 ModA murine GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGACCGCCCCCAGA- ACCCTGATCCTGCTG CD27L-CD40L CTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGCGGATCCCACCCCGAGCCCCACACCGCCGAAC- TGCAGCTGAACCTGA (DNA: CCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGCAGATCCTTTACACA- CGGCCCCGAGCTGGAAGAAGG 5'UTR- CCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCCTGGCCAACTGCAG- CAGCCCTGGCTCTACCCTGCAG CDS-3'UTR) CACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAGCCTGCTGAGAGGCAGATTCG- GCCAGGACTGTACCG TGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAACCTGACACTGCCTCTGCTGC- CCAGCAGAAACGCCGA CGAAACATTCTTTGGAGTGCAGTGGATTTGTCCTGGCGGAGGGTCCGGGGGAGGACACCCAGAACCTCATAC- AGCTGAACTGCAGCTG AACCTGACCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGCAGATCCTTTACA- CACGGCCCCGAGCTGG AAGAAGGCCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCCTGGCCAACTGCA- GCAGCCCTGGCTCTAC CCTGCAGCACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAGCCTGCTGAGAGG- CAGATTCGGCCAGGAC TGTACCGTGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAACCTGACACTGCCT- CTGCTGCCCAGCAGAA ACGCCGACGAAACATTCTTTGGAGTGCAGTGGATTTGTCCTGGGGGAGGCTCCGGAGGCGGACACCCTGAAC- CTCATACAGCTGAACT GCAGCTGAACCTGACCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGCAGATC- CTTTACACACGGCCCC GAGCTGGAAGAAGGCCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCCTGGCC- AACTGCAGCAGCCCTG GCTCTACCCTGCAGCACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAGCCTGC- TGAGAGGCAGATTCGG CCAGGACTGTACCGTGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAACCTGAC- ACTGCCTCTGCTGCCC AGCAGAAACGCCGACGAGACCTTCTTCGGCGTCCAGTGGATCTGCCCCGGAGGCGGTGGTAGTGGAGGTGGC- GGGTCCGGTGGAGGTG GAAGCGGCGACGAGGACCCCCAGATCGCCGCCCACGTGGTGTCTGAGGCCAACAGCAACGCCGCCTCTGTGC- TGCAGTGGGCCAAGAA AGGCTACTACACCATGAAGTCCAACCTCGTGATGCTGGAAAACGGCAAGCAGCTGACCGTGAAGCGCGAGGG- CCTGTACTATGTGTAC ACCCAAGTGACATTCTGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCTTTTATCGTGGGCCTGTGGCTGAAG- CCTAGCAGCGGCAGCG AGAGAATCCTGCTGAAGGCCGCCAACACCCACAGCAGCTCTCAGCTGTGCGAGCAGCAGTCTGTGCACCTGG- GAGGCGTGTTCGAGCT GCAAGCTGGCGCTTCCGTGTTCGTGAACGTGACCGAGGCCAGCCAAGTGATCCACAGAGTGGGCTTCAGCAG- CTTTGGACTGCTCAAA CTGGGCGGAGGGTCCGGCGGAGGCGGAGATGAAGATCCTCAGATTGCTGCCCACGTGGTGTCTGAGGCCAAC- AGCAACGCCGCCTCTG TGCTGCAGTGGGCCAAGAAAGGCTACTACACCATGAAGTCCAACCTCGTGATGCTGGAAAACGGCAAGCAGC- TGACCGTGAAGCGCGA GGGCCTGTACTATGTGTACACCCAAGTGACATTCTGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCTTTTAT- CGTGGGCCTGTGGCTG AAGCCTAGCAGCGGCAGCGAGAGAATCCTGCTGAAGGCCGCCAACACCCACAGCAGCTCTCAGCTGTGCGAG- CAGCAGTCTGTGCACC TGGGAGGCGTGTTCGAGCTGCAAGCTGGCGCTTCCGTGTTCGTGAACGTGACCGAGGCCAGCCAAGTGATCC- ACAGAGTGGGCTTCAG CAGCTTTGGACTGCTCAAACTGGGAGGCGGCTCCGGAGGCGGAGGAGATGAAGATCCTCAGATTGCTGCCCA- CGTGGTGTCTGAGGCC AACAGCAACGCCGCCTCTGTGCTGCAGTGGGCCAAGAAAGGCTACTACACCATGAAGTCCAACCTCGTGATG- CTGGAAAACGGCAAGC AGCTGACCGTGAAGCGCGAGGGCCTGTACTATGTGTACACCCAAGTGACATTCTGCAGCAACCGCGAGCCCA- GCAGCCAGAGGCCTTT TATCGTGGGCCTGTGGCTGAAGCCTAGCAGCGGCAGCGAGAGAATCCTGCTGAAGGCCGCCAACACCCACAG- CAGCTCTCAGCTGTGC GAGCAGCAGTCTGTGCACCTGGGAGGCGTGTTCGAGCTGCAAGCTGGCGCTTCCGTGTTCGTGAACGTGACC- GAGGCCAGCCAAGTGA TCCACAGAGTGGGCTTCTCCTCCTTCGGCCTCCTGAAGCTGTGACTCGAGAGCTCGCTTTCTTGCTGTCCAA- TTTCTATTAAAGGTTC CTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAA- TAAAAAACATTTATTT TCATTGCTGCGTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCA- ACTACTAAACTGGGGG ATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGAGAC- CTGGTCCAGAGTCGCT AGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA 74 ModA murine GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGACCGCCCCCAGA- ACCCUGAUCCUGCUG CD27L-CD40L CUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGCGGAUCCCACCCCGAGCCCCACACCGCCGAAC- UGCAGCUGAACCUGA (RNA) CCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGCAGAUCCUUUACACA- CGGCCCCGAGCUGGAAGAAGG CCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCCUGGCCAACUGCAGCAGCCC- UGGCUCUACCCUGCAG CACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAGCCUGCUGAGAGGCAGAUUC- GGCCAGGACUGUACCG UGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAACCUGACACUGCCUCUGCUGC- CCAGCAGAAACGCCGA CGAAACAUUCUUUGGAGUGCAGUGGAUUUGUCCUGGCGGAGGGUCCGGGGGAGGACACCCAGAACCUCAUAC- AGCUGAACUGCAGCUG AACCUGACCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGCAGAUCCUUUACA- CACGGCCCCGAGCUGG AAGAAGGCCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCCUGGCCAACUGCA- GCAGCCCUGGCUCUAC CCUGCAGCACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAGCCUGCUGAGAGG- CAGAUUCGGCCAGGAC UGUACCGUGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAACCUGACACUGCCU- CUGCUGCCCAGCAGAA ACGCCGACGAAACAUUCUUUGGAGUGCAGUGGAUUUGUCCUGGGGGAGGCUCCGGAGGCGGACACCCUGAAC- CUCAUACAGCUGAACU GCAGCUGAACCUGACCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGCAGAUC- CUUUACACACGGCCCC GAGCUGGAAGAAGGCCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCCUGGCC- AACUGCAGCAGCCCUG GCUCUACCCUGCAGCACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAGCCUGC- UGAGAGGCAGAUUCGG CCAGGACUGUACCGUGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAACCUGAC- ACUGCCUCUGCUGCCC AGCAGAAACGCCGACGAGACCUUCUUCGGCGUCCAGUGGAUCUGCCCCGGAGGCGGUGGUAGUGGAGGUGGC- GGGUCCGGUGGAGGUG GAAGCGGCGACGAGGACCCCCAGAUCGCCGCCCACGUGGUGUCUGAGGCCAACAGCAACGCCGCCUCUGUGC- UGCAGUGGGCCAAGAA AGGCUACUACACCAUGAAGUCCAACCUCGUGAUGCUGGAAAACGGCAAGCAGCUGACCGUGAAGCGCGAGGG- CCUGUACUAUGUGUAC ACCCAAGUGACAUUCUGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCUUUUAUCGUGGGCCUGUGGCUGAAG- CCUAGCAGCGGCAGCG AGAGAAUCCUGCUGAAGGCCGCCAACACCCACAGCAGCUCUCAGCUGUGCGAGCAGCAGUCUGUGCACCUGG- GAGGCGUGUUCGAGCU GCAAGCUGGCGCUUCCGUGUUCGUGAACGUGACCGAGGCCAGCCAAGUGAUCCACAGAGUGGGCUUCAGCAG- CUUUGGACUGCUCAAA CUGGGCGGAGGGUCCGGCGGAGGCGGAGAUGAAGAUCCUCAGAUUGCUGCCCACGUGGUGUCUGAGGCCAAC- AGCAACGCCGCCUCUG UGCUGCAGUGGGCCAAGAAAGGCUACUACACCAUGAAGUCCAACCUCGUGAUGCUGGAAAACGGCAAGCAGC- UGACCGUGAAGCGCGA GGGCCUGUACUAUGUGUACACCCAAGUGACAUUCUGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCUUUUAU- CGUGGGCCUGUGGCUG AAGCCUAGCAGCGGCAGCGAGAGAAUCCUGCUGAAGGCCGCCAACACCCACAGCAGCUCUCAGCUGUGCGAG- CAGCAGUCUGUGCACC UGGGAGGCGUGUUCGAGCUGCAAGCUGGCGCUUCCGUGUUCGUGAACGUGACCGAGGCCAGCCAAGUGAUCC- ACAGAGUGGGCUUCAG CAGCUUUGGACUGCUCAAACUGGGAGGCGGCUCCGGAGGCGGAGGAGAUGAAGAUCCUCAGAUUGCUGCCCA- CGUGGUGUCUGAGGCC AACAGCAACGCCGCCUCUGUGCUGCAGUGGGCCAAGAAAGGCUACUACACCAUGAAGUCCAACCUCGUGAUG- CUGGAAAACGGCAAGC AGCUGACCGUGAAGCGCGAGGGCCUGUACUAUGUGUACACCCAAGUGACAUUCUGCAGCAACCGCGAGCCCA- GCAGCCAGAGGCCUUU UAUCGUGGGCCUGUGGCUGAAGCCUAGCAGCGGCAGCGAGAGAAUCCUGCUGAAGGCCGCCAACACCCACAG- CAGCUCUCAGCUGUGC GAGCAGCAGUCUGUGCACCUGGGAGGCGUGUUCGAGCUGCAAGCUGGCGCUUCCGUGUUCGUGAACGUGACC- GAGGCCAGCCAAGUGA UCCACAGAGUGGGCUUCUCCUCCUUCGGCCUCCUGAAGCUGUGACUCGAGAGCUCGCUUUCUUGCUGUCCAA- UUUCUAUUAAAGGUUC CUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAA- UAAAAAACAUUUAUUU UCAUUGCUGCGUCGAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCA- ACUACUAAACUGGGGG AUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGUCGAGAC- CUGGUCCAGAGUCGCU AGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA 75 ModB murine MGAMAPRTLLLLLAAALAPTQTRAGPGSHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHL- RIHQDGLYRLHIQVT CD27L-CD40L LANCSSPGSTLQHRATLAVGICSPAAHGISLLRGRFGQDCTVALQRLTYLVHGDVLCTNLTLPLLPSRNADET- FFGVQWICPGGGSGG (amino GHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCS- SPGSTLQHRATLAVGICSPAAH acid) GISLLRGRFGQDCTVALQRLTYLVHGDVLCTNLTLPLLPSRNADETFFGVQWICPGGGSGGGHPEPH- TAELQLNLTVPRKDPTLRWGA GPALGRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCSSPGSTLQHRATLAVGICSPAAHGISLLRGRFGQ- DCTVALQRLTYLVHGD

VLCTNLTLPLLPSRNADETFFGVQWICPGGGGSGGGGSGGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKG- YYTMKSNLVMLENGKQ LTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQ- AGASVFVNVTEASQVI HRVGFSSFGLLKLGGGSGGGGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREG- LYYVYTQVTFCSNREP SSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSS- FGLLKLGGGSGGGGDE DPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFI- VGLWLKPSSGSERILL KAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL 76 ModB murine GGAATAAACTAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTG- CAGCAATTTAAATCA CD27L-CD40L TTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGCCATGGCCC- CTAGAACATTGCTCC (DNA: TGCTGCTGGCCGCTGCCCTGGCCCCTACACAGACAAGAGCTGGACCTGGATCCCACCCCGAGCCCCA- CACCGCCGAACTGCAGCTGAA 5'UTR- CCTGACCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGCAGATCCTT- TACACACGGCCCCGAGCTGGAA CDS-3'UTR) GAAGGCCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCCTGGCCAACTGCAGCA- GCCCTGGCTCTACCC TGCAGCACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAGCCTGCTGAGAGGCA- GATTCGGCCAGGACTG TACCGTGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAACCTGACACTGCCTCT- GCTGCCCAGCAGAAAC GCCGACGAAACATTCTTTGGAGTGCAGTGGATTTGTCCTGGCGGAGGGTCCGGGGGAGGACACCCAGAACCT- CATACAGCTGAACTGC AGCTGAACCTGACCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGCAGATCCT- TTACACACGGCCCCGA GCTGGAAGAAGGCCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCCTGGCCAA- CTGCAGCAGCCCTGGC TCTACCCTGCAGCACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAGCCTGCTG- AGAGGCAGATTCGGCC AGGACTGTACCGTGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAACCTGACAC- TGCCTCTGCTGCCCAG CAGAAACGCCGACGAAACATTCTTTGGAGTGCAGTGGATTTGTCCTGGGGGAGGCTCCGGAGGCGGACACCC- TGAACCTCATACAGCT GAACTGCAGCTGAACCTGACCGTGCCCAGAAAGGACCCCACCCTGAGATGGGGAGCTGGCCCTGCTCTGGGC- AGATCCTTTACACACG GCCCCGAGCTGGAAGAAGGCCACCTGAGAATCCACCAGGACGGCCTGTACAGACTGCACATCCAAGTGACCC- TGGCCAACTGCAGCAG CCCTGGCTCTACCCTGCAGCACAGAGCCACACTGGCCGTGGGCATCTGTAGCCCTGCTGCTCACGGAATCAG- CCTGCTGAGAGGCAGA TTCGGCCAGGACTGTACCGTGGCCCTGCAGAGGCTGACCTATCTGGTGCATGGCGACGTGCTGTGCACCAAC- CTGACACTGCCTCTGC TGCCCAGCAGAAACGCCGACGAGACCTTCTTCGGCGTCCAGTGGATCTGCCCCGGAGGCGGTGGTAGTGGAG- GTGGCGGGTCCGGTGG AGGTGGAAGCGGCGACGAGGACCCCCAGATCGCCGCCCACGTGGTGTCTGAGGCCAACAGCAACGCCGCCTC- TGTGCTGCAGTGGGCC AAGAAAGGCTACTACACCATGAAGTCCAACCTCGTGATGCTGGAAAACGGCAAGCAGCTGACCGTGAAGCGC- GAGGGCCTGTACTATG TGTACACCCAAGTGACATTCTGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCTTTTATCGTGGGCCTGTGGC- TGAAGCCTAGCAGCGG CAGCGAGAGAATCCTGCTGAAGGCCGCCAACACCCACAGCAGCTCTCAGCTGTGCGAGCAGCAGTCTGTGCA- CCTGGGAGGCGTGTTC GAGCTGCAAGCTGGCGCTTCCGTGTTCGTGAACGTGACCGAGGCCAGCCAAGTGATCCACAGAGTGGGCTTC- AGCAGCTTTGGACTGC TCAAACTGGGCGGAGGGTCCGGCGGAGGCGGAGATGAAGATCCTCAGATTGCTGCCCACGTGGTGTCTGAGG- CCAACAGCAACGCCGC CTCTGTGCTGCAGTGGGCCAAGAAAGGCTACTACACCATGAAGTCCAACCTCGTGATGCTGGAAAACGGCAA- GCAGCTGACCGTGAAG CGCGAGGGCCTGTACTATGTGTACACCCAAGTGACATTCTGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCT- TTTATCGTGGGCCTGT GGCTGAAGCCTAGCAGCGGCAGCGAGAGAATCCTGCTGAAGGCCGCCAACACCCACAGCAGCTCTCAGCTGT- GCGAGCAGCAGTCTGT GCACCTGGGAGGCGTGTTCGAGCTGCAAGCTGGCGCTTCCGTGTTCGTGAACGTGACCGAGGCCAGCCAAGT- GATCCACAGAGTGGGC TTCAGCAGCTTTGGACTGCTCAAACTGGGAGGCGGCTCCGGAGGCGGAGGAGATGAAGATCCTCAGATTGCT- GCCCACGTGGTGTCTG AGGCCAACAGCAACGCCGCCTCTGTGCTGCAGTGGGCCAAGAAAGGCTACTACACCATGAAGTCCAACCTCG- TGATGCTGGAAAACGG CAAGCAGCTGACCGTGAAGCGCGAGGGCCTGTACTATGTGTACACCCAAGTGACATTCTGCAGCAACCGCGA- GCCCAGCAGCCAGAGG CCTTTTATCGTGGGCCTGTGGCTGAAGCCTAGCAGCGGCAGCGAGAGAATCCTGCTGAAGGCCGCCAACACC- CACAGCAGCTCTCAGC TGTGCGAGCAGCAGTCTGTGCACCTGGGAGGCGTGTTCGAGCTGCAAGCTGGCGCTTCCGTGTTCGTGAACG- TGACCGAGGCCAGCCA AGTGATCCACAGAGTGGGCTTCTCCTCCTTCGGCCTCCTGAAGCTGTGACTCGACGTCCTGGTACTGCATGC- ACGCAATGCTAGCTGC CCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCC- CCACTCACCACCTCTG CTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGA- AACAGCAGTGATTAAC CTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC- CTCGAGCTAGCAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA 77 ModB murine GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUG- CAGCAAUUUAAAUCA CD27L-CD40L UUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCAUGGGCGCCAUGGCCC- CUAGAACAUUGCUCC (RNA) UGCUGCUGGCCGCUGCCCUGGCCCCUACACAGACAAGAGCUGGACCUGGAUCCCACCCCGAGCCCCA- CACCGCCGAACUGCAGCUGAA CCUGACCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGCAGAUCCUUUACACA- CGGCCCCGAGCUGGAA GAAGGCCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCCUGGCCAACUGCAGC- AGCCCUGGCUCUACCC UGCAGCACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAGCCUGCUGAGAGGCA- GAUUCGGCCAGGACUG UACCGUGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAACCUGACACUGCCUCU- GCUGCCCAGCAGAAAC GCCGACGAAACAUUCUUUGGAGUGCAGUGGAUUUGUCCUGGCGGAGGGUCCGGGGGAGGACACCCAGAACCU- CAUACAGCUGAACUGC AGCUGAACCUGACCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGCAGAUCCU- UUACACACGGCCCCGA GCUGGAAGAAGGCCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCCUGGCCAA- CUGCAGCAGCCCUGGC UCUACCCUGCAGCACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAGCCUGCUG- AGAGGCAGAUUCGGCC AGGACUGUACCGUGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAACCUGACAC- UGCCUCUGCUGCCCAG CAGAAACGCCGACGAAACAUUCUUUGGAGUGCAGUGGAUUUGUCCUGGGGGAGGCUCCGGAGGCGGACACCC- UGAACCUCAUACAGCU GAACUGCAGCUGAACCUGACCGUGCCCAGAAAGGACCCCACCCUGAGAUGGGGAGCUGGCCCUGCUCUGGGC- AGAUCCUUUACACACG GCCCCGAGCUGGAAGAAGGCCACCUGAGAAUCCACCAGGACGGCCUGUACAGACUGCACAUCCAAGUGACCC- UGGCCAACUGCAGCAG CCCUGGCUCUACCCUGCAGCACAGAGCCACACUGGCCGUGGGCAUCUGUAGCCCUGCUGCUCACGGAAUCAG- CCUGCUGAGAGGCAGA UUCGGCCAGGACUGUACCGUGGCCCUGCAGAGGCUGACCUAUCUGGUGCAUGGCGACGUGCUGUGCACCAAC- CUGACACUGCCUCUGC UGCCCAGCAGAAACGCCGACGAGACCUUCUUCGGCGUCCAGUGGAUCUGCCCCGGAGGCGGUGGUAGUGGAG- GUGGCGGGUCCGGUGG AGGUGGAAGCGGCGACGAGGACCCCCAGAUCGCCGCCCACGUGGUGUCUGAGGCCAACAGCAACGCCGCCUC- UGUGCUGCAGUGGGCC AAGAAAGGCUACUACACCAUGAAGUCCAACCUCGUGAUGCUGGAAAACGGCAAGCAGCUGACCGUGAAGCGC- GAGGGCCUGUACUAUG UGUACACCCAAGUGACAUUCUGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCUUUUAUCGUGGGCCUGUGGC- UGAAGCCUAGCAGCGG CAGCGAGAGAAUCCUGCUGAAGGCCGCCAACACCCACAGCAGCUCUCAGCUGUGCGAGCAGCAGUCUGUGCA- CCUGGGAGGCGUGUUC GAGCUGCAAGCUGGCGCUUCCGUGUUCGUGAACGUGACCGAGGCCAGCCAAGUGAUCCACAGAGUGGGCUUC- AGCAGCUUUGGACUGC UCAAACUGGGCGGAGGGUCCGGCGGAGGCGGAGAUGAAGAUCCUCAGAUUGCUGCCCACGUGGUGUCUGAGG- CCAACAGCAACGCCGC CUCUGUGCUGCAGUGGGCCAAGAAAGGCUACUACACCAUGAAGUCCAACCUCGUGAUGCUGGAAAACGGCAA- GCAGCUGACCGUGAAG CGCGAGGGCCUGUACUAUGUGUACACCCAAGUGACAUUCUGCAGCAACCGCGAGCCCAGCAGCCAGAGGCCU- UUUAUCGUGGGCCUGU GGCUGAAGCCUAGCAGCGGCAGCGAGAGAAUCCUGCUGAAGGCCGCCAACACCCACAGCAGCUCUCAGCUGU- GCGAGCAGCAGUCUGU GCACCUGGGAGGCGUGUUCGAGCUGCAAGCUGGCGCUUCCGUGUUCGUGAACGUGACCGAGGCCAGCCAAGU- GAUCCACAGAGUGGGC UUCAGCAGCUUUGGACUGCUCAAACUGGGAGGCGGCUCCGGAGGCGGAGGAGAUGAAGAUCCUCAGAUUGCU- GCCCACGUGGUGUCUG AGGCCAACAGCAACGCCGCCUCUGUGCUGCAGUGGGCCAAGAAAGGCUACUACACCAUGAAGUCCAACCUCG- UGAUGCUGGAAAACGG CAAGCAGCUGACCGUGAAGCGCGAGGGCCUGUACUAUGUGUACACCCAAGUGACAUUCUGCAGCAACCGCGA- GCCCAGCAGCCAGAGG CCUUUUAUCGUGGGCCUGUGGCUGAAGCCUAGCAGCGGCAGCGAGAGAAUCCUGCUGAAGGCCGCCAACACC- CACAGCAGCUCUCAGC UGUGCGAGCAGCAGUCUGUGCACCUGGGAGGCGUGUUCGAGCUGCAAGCUGGCGCUUCCGUGUUCGUGAACG- UGACCGAGGCCAGCCA AGUGAUCCACAGAGUGGGCUUCUCCUCCUUCGGCCUCCUGAAGCUGUGACUCGACGUCCUGGUACUGCAUGC- ACGCAAUGCUAGCUGC CCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCC- CCACUCACCACCUCUG CUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGA- AACAGCAGUGAUUAAC CUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC- CUCGAGCUAGCAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA Other sequences of the invention 78 Poly-A AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAA 79 Anti-PD1 EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY Mab heavy LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS chain GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 80 Anti-PD1 DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLLIYA ASSLHGGVPS RFSGSGSGTD FTLTIRTLQP Mab light EDFATYYCQQ SSNTPFTFGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ chain ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 81 HCDR1 GFTFSNFG 82 HCDR2 ISGGGRDT 83 HCDR3 VKWGNIYFDY 84 LCDR1 LSINTF 85 LCDR2 AAS 86 LCDR3 QQSSNTPFT 87 Anti-PD1 EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY Mab VH LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSS 88 Anti-PD1 DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLLIYA ASSLHGGVPS RFSGSGSGTD FTLTIRTLQP Mab VL EDFATYYCQQ SSNTPFTFGP GTVVDFR

DETAILED DESCRIPTION

I. Definitions

[0595] The term "ModB" describes RNA comprising a modified nucleobase in place of at least one (e.g., every) uridine and further comprising a Cap1 structure at the 5' end of the RNA. In some embodiments, the 5' UTR of a ModB RNA comprises SEQ ID NOs: 4 or 6. ModB RNA has been processed to reduce double-stranded RNA (dsRNA). The "Cap1" structure may be generated after in-vitro translation by enzymatic capping or during in-vitro translation (co-transcriptional capping).

[0596] In some embodiments, the building block cap for ModB modified RNA is as follows, which is used when co-transcriptionally capping:

m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG (also sometimes referred to as m.sub.2.sup.7,3'OG(5')ppp(5')m.sup.2'-OApG), which has the following structure:

##STR00001##

[0597] Below is an exemplary Cap1 RNA after co-transcriptional capping, which comprises RNA and m.sub.2.sup.7,3'OG(5')ppp(5')m.sup.2'-OApG:

##STR00002##

[0598] Below is another exemplary Cap1 RNA after enzymatic capping (no cap analog):

##STR00003##

[0599] The term "ModA" describes RNA without dsRNA reduction that does not comprise a modified nucleobase in place of at least one uridine. ModA RNA comprises a Cap0 structure at the 5' end of the RNA. The 5' UTR of a ModA RNA may comprise SEQ ID NO: 2. "Cap0" structures are generated during in-vitro translation (co-transcriptional capping) using, in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m.sub.2.sup.7,3'OG(5')ppp(5')G)) with the structure:

##STR00004##

[0600] Below is an exemplary Cap0 RNA comprising RNA and m.sub.2.sup.7,3'OG(5')ppp(5')G:

##STR00005##

[0601] In some embodiments, the "Cap0" structures are generated during in-vitro translation (co-transcriptional capping) using the cap analog Beta-S-ARCA (m.sub.2.sup.7,2'OG(5')ppSp(5')G) with the structure:

##STR00006##

[0602] Below is an exemplary Cap0 RNA comprising Beta-S-ARCA (m.sub.2.sup.7,2'OG(5')ppSp(5')G) and RNA.

##STR00007##

[0603] The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

##STR00008##

[0604] The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

##STR00009##

[0605] UTP (uridine 5'-triphosphate) has the following structure:

##STR00010##

[0606] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

##STR00011##

[0607] "Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Pseudouridine is described, for example, in Charette and Gray, Life; 49:341-351 (2000).

[0608] Another exemplary modified nucleoside is N1-methylpseudouridine (m1.PSI.), which has the structure:

##STR00012##

[0609] N1-Methylpseudo-UTP has the following structure:

##STR00013##

[0610] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule. Poly-A tails or poly-A sequences are known to those of skill in the art, and may follow the 3' UTR in the RNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs disclosed herein can have a poly-A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0611] It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0612] The poly-A tail may be of any length. In one embodiment, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context "essentially consists of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0613] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0614] In one embodiment of the present invention, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in the present invention. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency. Consequently, in one embodiment of the present invention, the poly-A tail contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0615] In one embodiment of the invention, no nucleotides other than A nucleotides flank a poly-A tail at its 3' end, i.e., the poly-A tail is not masked or followed at its 3' end by a nucleotide other than A.

[0616] In some embodiments, a poly-A tail comprises the sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID NO: 78), which is also shown in Table 2 following the 3' UTR sequence.

[0617] "RNA" and "mRNA" are used interchangeably herein.

[0618] "IFN.alpha." is used generically herein to describe any interferon alpha Type I cytokine, including IFN.alpha.2b and IFN.alpha.4. In the experiments described in the Example section, human IFN.alpha.2b and mouse IFN.alpha.4 were utilized. Any IFN.alpha. may be incorporated into the compositions and used in the methods described herein.

[0619] The term "treatment," as used herein, covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of the disease. For example, treatment of a solid tumor may comprise alleviating symptoms of the solid tumor, decreasing the size of the solid tumor, eliminating the solid tumor, reducing further growth of the tumor, or reducing or eliminating recurrence of a solid tumor after treatment. Treatment may also be measured as a change in a biomarker of effectiveness or in an imaging or radiographic measure.

[0620] The term "prevention," as used herein, means inhibiting or arresting development of cancer, including solid tumors, in a subject deemed to be cancer free.

[0621] "Metastasis" means the process by which cancer spreads from the place at which it first arose as a primary tumor to other locations in the body.

[0622] The term "intra-tumorally," as used herein, means into the tumor. For example, intra-tumoral injection means injecting the therapeutic at any location that touches the tumor.

[0623] The term "peri-tumorally," or "peri-tumoral," as used herein, is an area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery. The peri-tumoral area comprises host tissue. See, for example, FIG. 36.

[0624] "Administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

[0625] The disclosure describes nucleic acid sequences and amino acid sequences having a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).

[0626] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0627] The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0628] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0629] In some embodiments, the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0630] Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence. One important property includes the ability to act as a cytokine, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to said given sequence.

II. Compositions and Medical Preparations

[0631] A. Interleukin-2 (IL-2)

[0632] In some embodiments, the composition comprises a DNA sequence encoding interleukin-2 (IL-2) (SEQ ID NO: 9). In some embodiments, the DNA sequence encoding IL-2 is provided in SEQ ID NO: 10.

[0633] In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-2. In some embodiments, the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NOs: 11. In some embodiments, the DNA sequence comprises a codon-optimized DNA sequence with 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11.

[0634] The alignment of codon optimized IL-2 to native IL-2 is shown below, where the "Q" is native IL-2 (NM_000586.3; SEQ ID NO: 10) and the "S" is codon optimized IL-2 (SEQ ID NO: 11). The percent identity is 82.79%.

TABLE-US-00003 Q: 1 ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT 60 |||||||| ||||| || |||||||||||||| || |||||| ||||| ||||| | S: 1 ATGTACAGAATGCAGCTGCTGTCTTGCATTGCTCTTTCTCTTGCTCTTGTGACAAATTCT 60 Q: 61 GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGAT 120 || || || || ||| ||||||||||||||||| || || || || | || || ||| S: 61 GCTCCAACATCTTCTTCAACAAAGAAAACACAGCTTCAGCTTGAACACCTTCTTCTTGAT 120 Q: 121 TTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTC 180 | ||||||||| |||||||||| || |||||||| ||||| ||||| || || ||||| S: 121 CTTCAGATGATTCTGAATGGAATCAACAATTACAAAAATCCAAAACTGACAAGAATGCTG 180 Q: 181 ACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAA 240 |||||||| ||||||||||| ||||| || |||||||||||||| |||||||| || ||| S: 181 ACATTTAAATTTTACATGCCAAAGAAAGCAACAGAACTGAAACACCTTCAGTGCCTTGAA 240 Q: 241 GAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTA 300 |||||||| ||||||||||| |||||||| ||| | ||||| |||||||| |||||| | S: 241 GAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTA 300 Q: 301 AGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAA 360 ||||| || || | |||||||| ||||| || || || |||||||| || ||||||||| S: 301 AGACCAAGAGATCTGATCAGCAACATCAATGTGATTGTGCTGGAACTGAAAGGATCTGAA 360 Q: 361 ACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGA 420 ||||||||||||||||||||||||||||| |||||||| ||||| ||||||||||||||| S: 361 ACAACATTCATGTGTGAATATGCTGATGAAACAGCAACAATTGTGGAATTTCTGAACAGA 420 Q: 421 TGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACT 459 ||||| || ||||| || ||||| ||||||||||| S: 421 TGGATCACATTTTGCCAGTCAATCATTTCAACACTGACA 459

[0635] In some embodiments, the composition comprises an RNA sequence transcribed from a DNA sequence encoding IL-2. In some embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 10 or 11. In some embodiments, the RNA sequence comprises or consists of SEQ ID NOs: 12 or 13. In some embodiments, the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 12 or 13.

[0636] In some embodiments, one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0637] In some embodiments, the IL-2 RNA comprises an altered nucleotide at the 5' end. In some embodiments, the IL-2 RNA comprises a 5' cap. Any 5' cap known in the art may be used. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage including thiophosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3'-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5' cap comprises 7-methylguanylate. In some embodiments, the 5' cap is Cap0 or Cap1. Exemplary cap structures include m7G(5')ppp(5')G, m7,2' O-mG(5')ppsp(5')G, m7G(5')ppp(5')2' O-mG, and m7,3' O-mG(5')ppp(5')2' O-mA.

[0638] In some embodiments, the IL-2 RNA comprises a 5' untranslated region (UTR). In some embodiments, the 5' UTR is upstream of the initiation codon. In some embodiments, the 5' UTR regulates translation of the RNA. In some embodiments, the 5' UTR is a stabilizing sequence. In some embodiments, the 5' UTR increases the half-life of RNA. Any 5' UTR known in the art may be used. In some embodiments, the 5' UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 1, 3, or 5. In some embodiments, the 5' UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6. In some embodiments, the 5' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.

[0639] In some embodiments, the IL-2 RNA comprises a 3' UTR. In some embodiments, the 3' UTR follows the translation termination codon. In some embodiments, the 3' UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA. In some embodiments, the 3' UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 7. In some embodiments, the 3' UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0640] In some embodiments, the IL-2 composition comprises both a 5' UTR and a 3' UTR. In some embodiments, the composition comprises only a 5' UTR. In some embodiments, the composition comprises only a 3' UTR.

[0641] In some embodiments, the IL-2 RNA comprises a poly-A tail. In some embodiments, the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50, at least about 70, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.

[0642] In some embodiments, the RNA comprises a 5' cap, a 5' UTR, a nucleic acid encoding IL-2, a 3' UTR, and a poly-A tail, in that order.

[0643] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.

[0644] In some embodiments, the composition comprises an RNA sequence, that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0645] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0646] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0647] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0648] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced.

[0649] In some embodiments, one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0650] In some embodiments, the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 12 or 13; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0651] B. Interleukin-12 Single-Chain (IL-12sc)

[0652] In some embodiments, the composition comprises a DNA sequence encoding interleukin-12 single-chain (IL-12sc) (e.g., SEQ ID NO: 14), which comprises IL-12 p40 (sometimes referred to as IL-12B), a linker, such as a GS linker, and IL-12 p35 (sometimes referred to as IL-12A). In some embodiments, the IL-12p40, linker, and IL-12p35 are consecutive with no intervening nucleotides. An exemplary DNA sequence encoding IL-12sc is provided in SEQ ID NO: 15.

[0653] The alignment of codon optimized IL-12 p40 to native IL-12 p40 is shown below, where the "S" is native IL-12 p40 (NM_002187.2; nucleotides 1-984 of SEQ ID NO: 15) and the "Q" is codon optimized IL-12 p40 (nucleotides 1-984 of SEQ ID NO: 16). The percent identity is 77%.

TABLE-US-00004 Q: 1 ATGTGTCACCAGCAGCTGGTGATCTCATGGTTCTCCCTGGTATTTCTGGCATCTCCTCTT 60 ||||||||||||||| |||| ||||| ||||| |||||||| |||||||||||||| ||| S: 1 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTC 60 Q: 61 GTCGCAATCTGGGAACTGAAGAAAGACGTGTATGTCGTTGAGCTCGACTGGTATCCGGAT 120 || || || ||||||||||||||||| || |||||||| || | || |||||||||||| S: 61 GTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGAT 120 Q: 121 GCGCCTGGCGAGATGGTGGTGCTGACCTGTGACACCCCAGAGGAGGATGGGATCACTTGG 180 || ||||| || |||||||| || |||||||||||||| || || ||||| ||||| ||| S: 121 GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGG 180 Q: 181 ACCCTTGATCAATCCTCCGAAGTGCTCGGGTCTGGCAAGACTCTGACCATACAAGTGAAA 240 ||| | || || | || || | || |||||||| || |||||||| ||||| ||| S: 181 ACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAA 246 Q: 241 GAGTTTGGCGATGCCGGGCAGTACACTTGCCATAAGGGCGGAGAAGTTCTGTCCCACTCA 300 |||||||| ||||| || |||||||| || || || || || || ||||| ||| || S: 241 GAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCG 300 Q: 301 CTGCTGCTGCTGCACAAGAAAGAGGACGGAATTTGGAGTACCGATATCCTGAAAGATCAG 360 || |||||||| ||||| || || || ||||||||| || ||||| | || || ||| S: 301 CTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAG 360 Q: 361 AAAGAGCCCAAGAACAAAACCTTCTTGCGGTGCGAAGCCAAGAACTACTCAGGGAGATTT 420 ||||| ||||| || || ||||| | | ||||| |||||||| || || || | || S: 361 AAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTC 420 Q: 421 ACTTGTTGGTGGCTGACGACGATCAGCACCGATCTGACTTTCTCCGTGAAATCAAGTAGG 480 || || |||||||||||||| ||||| || ||| |||| ||| || ||| || || S: 421 ACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGA 480 Q: 481 GGATCATCTGACCCTCAAGGAGTCACATGTGGAGCGGCTACTCTGAGCGCTGAACGCGTA 540 || || |||||||| ||||| || || || ||||| ||||| || || || | || S: 481 GGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTC 540 Q: 541 AGAGGGGACAATAAGGAGTACGAGTATAGCGTTGAGTGCCAAGAGGATAGCGCATGCCCC 690 ||||||||||| |||||||| ||||| || |||||||| ||||| || || ||||| S: 541 AGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCA 690 Q: 601 GCCGCCGA--AGAATCATTGCCCATTGAAGTGATGGTGGATGCTGTACACAAGCTGAAGT 658 || || || ||| || | ||||||||| || ||||||||||| || |||||||| |||| S: 601 GCTGCTGAGGAGAGTC-T-GCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGT 858 Q: 659 ATGAGAACTACACAAGCTCCTTCTTCATCCGTGACATCATCAAACCAGATCCTCCTAAGA 718 |||| |||||||| ||| |||||||||| | |||||||||||||| || || || |||| S: 659 ATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGA 718 Q: 719 ACCTCCAGCTTAAACCTCTGAAGAACTCTAGACAGGTGGAAGTGTCTTGGGAGTATCCCG 778 || | ||||| || || | ||||| ||| | |||||||| || |||||||| || | S: 719 ACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTG 778 Q: 779 ACACCTGGTCTACACCACATTCCTACTTCAGTCTCACATTCTGCGTTCAGGTACAGGGCA 838 |||||||| ||| ||||||||||||||| || ||||||||||||||||| ||||||| S: 779 ACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCA 838 Q: 839 AGTCCAAAAGGGAGAAGAAGGATCGGGTCTTTACAGATAAAACAAGTGCCACCGTTATAT 898 || ||| || || ||||| ||| | ||||| || || || || ||||| || || | S: 839 AGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCT 898 Q: 899 GCCGGAAGAATGCCTCTATTTCTGTGCGTGCGCAGGACAGATACTATAGCAGCTCTTGGA 958 |||| || |||||| ||| ||||| || |||||| | ||||||||| ||||||| S: 899 GCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGA 958 Q 959 GTGAATGGGC--CAGTGTCCCATGTTCA 984 | |||||||| | ||| ||| || || S: 959 GCGAATGGGCATCTGTG-CCC-TG--CA 982

[0654] The alignment of codon optimized IL-12 p35 to native IL-12 p35 is shown below, where the "S" is native IL-12 p35 (NM_00882.3; nucleotides 1027-1623 of SEQ ID NO: 15) and the "Q" is codon optimized IL-12 p35 (nucleotides 1027-1623 of SEQ ID NO: 16). The percent identity is 80%.

TABLE-US-00005 Q: 1 AGAAATCTCCCTGTGGCTACACCTGATCCAGGCATGTTTCCCTGTTTGCACCATAGCCAA 60 ||||| ||||| ||||| || || || ||||| ||||| || || | ||||| |||| S: 1 AGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA 60 Q: 61 AACCTCCTGAGAGCAGTCAGCAACATGCTCCAGAAAGCTAGACAAACACTGGAATTCTAC 120 ||||| ||||| || |||||||||||||||||||| || |||||||| || ||||| ||| S: 61 AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTAC 120 Q: 121 CCATGCACCTCCGAGGAAATAGATCACGAGGATATCACTAAGGACAAAACAAGCACTGTC 180 || ||||| || || || || ||||| || |||||||| || || ||||| ||||| || S: 121 CCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTG 180 Q. 181 GAAGCATGCCTTCCCTTGGAACTGACAAAGAACGAGAGTTGCCTTAATTCAAGAGAAACA 240 || || || | || |||||| | || ||||| ||||||||||| ||||| ||||| || S: 181 GAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACC 240 Q: 241 TCTTTCATTACAAACGGTAGCTGCTTGGCAAGCAGAAAAACATCTTTTATGATGGCCCTT 300 |||||||| || || || || ||| |||| |||||| || ||||||||||||||||| S 241 TCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTG 300 Q: 301 TGTCTGAGCAGTATTTATGAGGATCTCAAAATGTACCAGGTGGAGTTTAAGACCATGAAT 360 || || || || |||||||| || | || ||||||||||||||||| ||||||||||| S: 301 TGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAAT 360 Q: 361 GCCAAGCTGCTGATGGACCCAAAGAGACAGATTTTCCTCGATCAGAATATGCTGGCTGTG 420 || ||||| |||||||| || ||||| ||||| || || ||||| || |||||||| || S: 361 GCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTT 420 Q: 421 ATTGATGAACTGATGCAGGCCTTGAATTTCAACAGCGAAACCGTTCCCCAGAAAAGCAGT 480 |||||||| |||||||||||| ||||||||||||| || || || || || ||| | S: 421 ATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCC 480 Q: 481 CTTGAAGAACCTGACTTTTATAAGACCAAGATCAAACTGTGTATTCTCCTGCATGCCTTT 540 ||||||||||| || |||||||| || || ||||| || || || || || ||||| || S: 401 CTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTC 540 Q: 541 AGAATCAGAGCAGTCACTATAGATAGAGTGATGTCCTACCTGAATGCTTCC 591 ||||| | ||||| ||||| |||||||||||| ||| |||||||||||| S: 541 AGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCC 591

[0655] In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-12sc. In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-12 p40. In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-12 p35. In some embodiments, the codon-optimized DNA sequence comprises or consists of SEQ ID NO: 16. In some embodiments, the DNA sequence comprises a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16. In some embodiments, the codon-optimized DNA sequence encoding IL-12 p40 comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16). In some embodiments, the codon-optimized DNA sequence encoding IL-12 p35 comprises the nucleotides encoding the IL-12sc-p35 (nucleotides 1027-1623 of SEQ ID NO: 16). In some embodiments, the codon-optimized DNA sequence encoding IL-12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16) and -p35 (nucleotides 1027-1623 of SEQ ID NO: 16) portions of SEQ ID NO: 16 and further comprises nucleotides between the p40 and p35 portions (e.g., nucleotides 985-1026 of SEQ ID NO: 16) encoding a linker polypeptide connecting the p40 and p35 portions. Any linker known to those of skill in the art may be used. The p40 portion may be 5' or 3' to the p35 portion.

[0656] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-12sc. The RNA may also be recombinantly produced. In some embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 15 or 16. In some embodiments, the RNA sequence comprises or consists of SEQ ID NOs: 17 or 18. In some embodiments, the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 17 or 18. In some embodiments, the RNA sequence comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NOs: 17 or 18) and -p35 (nucleotides 1027-1623 of SEQ ID NOs: 17 or 18) portions of SEQ ID NOs: 17 or 18. In some embodiments, the codon-optimized RNA sequence encoding IL-12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 18) and -p35 (nucleotides 1027-1623 of SEQ ID NO: 18) portions of SEQ ID NO: 18 and further comprises nucleotides between the p40 and p35 portions encoding a linker polypeptide connecting the p40 and p35 portions. Any linker known to those of skill in the art may be used.

[0657] In some embodiments, one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0658] In some embodiments, the IL-12sc RNA comprises an altered nucleotide at the 5' end. In some embodiments, the RNA comprises a 5' cap. Any 5' cap known in the art may be used. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage including thiophosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3'-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5' cap comprises 7-methylguanylate. In some embodiments, the 5' cap is Cap0 or Cap1. Exemplary cap structures include m7G(5')ppp(5')G, m7,2' O-mG(5')ppsp(5')G, m7G(5')ppp(5')2' O-mG, and m7,3' O-mG(5')ppp(5')2' O-mA.

[0659] In some embodiments, the IL-12sc RNA comprises a 5' untranslated region (UTR). In some embodiments, the 5' UTR is upstream of the initiation codon. In some embodiments, the 5' UTR regulates translation of the RNA. In some embodiments, the 5' UTR is a stabilizing sequence. In some embodiments, the 5' UTR increases the half-life of RNA. Any 5' UTR known in the art may be used. In some embodiments, the 5' UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5. In some embodiments, the 5' UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6. In some embodiments, the 5' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.

[0660] In some embodiments, the IL-12sc RNA comprises a 3' UTR. In some embodiments, the 3' UTR follows the translation termination codon. In some embodiments, the 3' UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA. In some embodiments, the 3' UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3' UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0661] In some embodiments, the IL-12sc composition comprises both a 5' UTR and a 3' UTR. In some embodiments, the IL-12sc composition comprises only a 5' UTR. In some embodiments, the IL-12sc composition comprises only a 3' UTR.

[0662] In some embodiments, the IL-12sc RNA comprises a poly-A tail. In some embodiments, the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.

[0663] In some embodiments, the RNA comprises a 5' cap, a 5' UTR, a nucleic acid encoding IL-12sc, a 3' UTR, and a poly-A tail, in that order.

[0664] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.

[0665] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0666] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0667] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0668] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0669] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0670] In some embodiments, the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 17 or 18; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0671] C. Interferon Alpha (IFN.alpha.)

[0672] In some embodiments, the composition comprises a DNA sequence encoding interferon alpha (IFN.alpha.) (e.g., SEQ ID NO: 19). An exemplary DNA sequence encoding this IFN.alpha. is provided in SEQ ID NO: 20.

[0673] The alignment of codon optimized IFN.alpha. to native IFN.alpha. is shown below, where the "S" is native IFN.alpha. (NM_000605.3; SEQ ID NO: 20) and the "Q" is codon optimized IFN.alpha. (SEQ ID NO: 21). The percent identity is 79%.

TABLE-US-00006 Q 1 ATGGCCCTGACTTTTGCCCTTCTCGTGGCTTTGTTGGTGCTGAGTTGCAAATCTTCCTGT 60 |||||| |||| ||||| | || ||||| | ||||||| || ||||| || ||| S: 1 ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGC 60 Q: 61 AGTGTCGGATGTGATCTGCCTCAAACCCACAGTCTGGG-ATCTAGGAGAACACTGATGCT 119 ||| || ||||||||||||||||||||||| ||||| | | ||||| || ||||||| S: 61 TCTGTGGGCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGC-AGGAGGACCTTGATGCT 119 Q: 120 GTTGGCACAGATGAGGAGAAT-TAGC-CTCTTTTCCTGCCTGAAGGATAGACATGACTTC 177 ||||||||||||||||||| | | || || |||||| ||||||| ||||||||||| S: 120 CCTGGCACAGATGAGGAGAATCT--CTCTTTTCTCCTGCTTGAAGGACAGACATGACTTT 177 Q: 178 GGCTTTCCCCAAGAGGAGTTTGGCAATCAGTTCCAGAAAGCGGAAACGATTCCCGTTCTG 237 || |||||||| |||||||||||||| |||||||| || || ||||| || || || || S: 178 GGATTTCCCCAGGAGGAGTTTGGCAACCAGYYCCAAAAGGCTGAAACCATCCCTGTCCTC 237 Q: 238 CACGAGATGATCCAGCAGATCTTCAACCTCTTTTCAAC-CAAAG-ACAGCTCAGCAGCCT 295 || ||||||||||||||||||||||| ||||| || | ||||| || || || || | S: 238 CATGAGATGATCCAGCAGATCTTCAATCTCTT--CAGCACAAAGGACTCATCTGCTGCTT 295 Q: 296 GGGATGAGACACTGCTGGACAAATTCTACACAGAACTGTATCAGCAGCTTAACGATCTGG 355 |||||||||| || || |||||||||||||| ||||| || |||||||| || || |||| S: 296 GGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGG 355 Q 356 AGGCATGCGTGATCCAAGGGGTTGGTGTGACTGAAACTCCGCTTATGAAGGAGGACTCCA 415 | || || ||||| || ||||| || ||||| || ||||| || |||||||||||||||| S: 356 AAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCA 415 Q: 416 TTCTGGCTGTACGGAAGTACTTCCAGAGAATAACCCTCTATCTGAAGGAGAAGAAGTACT 475 |||||||||| |||| |||||||| ||||| || ||||||||||| |||||||| ||| S: 416 TTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACA 475 Q: 476 CACCATGTGCTTGGGAAGTCGTGAGAGCCGAAATCATGAGATCCTTCAGCCTTAG-CACC 534 || ||||| ||||| || || ||||| |||||||||||||| || | || | || | S: 476 GCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATC-TTTTTC-TTTGTCAAC 533 Q: 535 AATC-TCCAGGAATCTCTGAGAAGCAAAGAG 564 || | | || ||| | | ||||| || || S: 534 AAACTTGCAAGAAAGTTTAAGAAGTAAGGAA 564

[0674] In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IFN.alpha.. In some embodiments, the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NO: 21. In some embodiments, the DNA sequence comprises or consists of a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0675] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IFN.alpha.. The RNA may also be recombinantly produced. In some embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 20 or 21. In some embodiments, the RNA sequence comprises or consists of SEQ ID NOs: 22 or 23. In some embodiments, the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22 or 23.

[0676] In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, each uridine in the RNA is modified. In some embodiments, each uridine in the RNA is modified with N1-methyl-pseudouridine (m.sup.1.psi.).

[0677] In some embodiments, the IFN.alpha. RNA comprises an altered nucleotide at the 5' end. In some embodiments, the IFN.alpha. RNA comprises a 5' cap. Any 5' cap known in the art may be used. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage including thiophosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3''-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5' cap comprises 7-methylguanylate. In some embodiments, the 5' cap is Cap0 or Cap1. Exemplary cap structures include m7G(5')ppp(5')G, m7,2' O-mG(5')ppsp(5')G, m7G(5')ppp(5')2' O-mG and m7,3' O-mG(5')ppp(5')2' O-mA.

[0678] In some embodiments, the IFN.alpha. RNA comprises a 5' untranslated region (UTR). In some embodiments, the 5' UTR is upstream of the initiation codon. In some embodiments, the 5' UTR regulates translation of the RNA. In some embodiments, the 5' UTR is a stabilizing sequence. In some embodiments, the 5' UTR increases the half-life of RNA. Any 5' UTR known in the art may be used. In some embodiments, the 5' UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 1, 3, or 5. In some embodiments, the 5' UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6. In some embodiments, the 5' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.

[0679] In some embodiments, the IFN.alpha. RNA comprises a 3' UTR. In some embodiments, the 3' UTR follows the translation termination codon. In some embodiments, the 3' UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA. In some embodiments, the 3' UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 7. In some embodiments, the 3' UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0680] In some embodiments, the IFN.alpha. composition comprises both a 5' UTR and a 3' UTR. In some embodiments, the composition comprises only a 5' UTR. In some embodiments, the composition comprises only a 3' UTR.

[0681] In some embodiments, the IFN.alpha. RNA comprises a poly-A tail. In some embodiments, the IFN.alpha. RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.

[0682] In some embodiments, the RNA comprises a 5' cap, a 5' UTR, a nucleic acid encoding IFN.alpha., a 3' UTR, and a poly-A tail, in that order.

[0683] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.

[0684] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0685] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0686] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0687] In some embodiments, the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0688] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.). In some embodiments, the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 22 or 23; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0689] D. IL-15 Sushi

[0690] As used herein, the term "IL-15 sushi" describes a construct comprising the soluble interleukin 15 (IL-15) receptor alpha sushi domain and mature interleukin alpha (IL-15) as a fusion protein. In some embodiments, the composition comprises a DNA sequence encoding IL-15 sushi (SEQ ID NO: 24), which comprises the soluble IL-15 receptor alpha chain (sushi) followed by a glycine-serine (GS) linker followed by the mature sequence of IL-15. The DNA sequence encoding this IL-15 sushi is provided in SEQ ID NO: 25.

[0691] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-15 sushi. The RNA may also be recombinantly produced. In some embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 25. In some embodiments, the nucleotides encoding the linker may be completely absent or replaced in part or in whole with any nucleotides encoding a suitable linker. In some embodiments, the RNA sequence comprises or consists of SEQ ID NO: 26. In some embodiments, the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26. In some embodiments, the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g., nucleotide 382-729 of SEQ ID NO: 25 or 26). In some embodiments, the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g., nucleotide 382-729 of SEQ ID NOs: 25 or 26) and further comprises nucleotides between these portions encoding a linker polypeptide connecting the portions. In some embodiments, the linker comprises nucleotides 322-381 of SEQ ID Nos: 25 or 26. Any linker known to those of skill in the art may be used.

[0692] In some embodiments, one or more uridine in the IL-15 sushi RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0693] In some embodiments, the IL-15 sushi RNA comprises an altered nucleotide at the 5' end. In some embodiments, the IL-15 sushi RNA comprises a 5' cap. Any 5' cap known in the art may be used. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage including thiophosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3'-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5' cap comprises 7-methylguanylate. In some embodiments, the 5' cap is Cap0 or Cap1. Exemplary cap structures include m7G(5')ppp(5')G, m7,2' O-mG(5')ppsp(5')G, m7G(5')ppp(5')2' O-mG and m7,3' O-mG(5')ppp(5')2' O-mA.

[0694] In some embodiments, the IL-15 sushi RNA comprises a 5' untranslated region (UTR). In some embodiments, the 5' UTR is upstream of the initiation codon. In some embodiments, the 5' UTR regulates translation of the RNA. In some embodiments, the 5' UTR is a stabilizing sequence. In some embodiments, the 5' UTR increases the half-life of RNA. Any 5' UTR known in the art may be used. In some embodiments, the 5' UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5. In some embodiments, the 5' UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6. In some embodiments, the 5' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.

[0695] In some embodiments, the IL-15 sushi RNA comprises a 3' UTR. In some embodiments, the 3' UTR follows the translation termination codon. In some embodiments, the 3' UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA. In some embodiments, the 3' UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3' UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0696] In some embodiments, the IL-15 sushi composition comprises both a 5' UTR and a 3' UTR. In some embodiments, the IL-15 sushi composition comprises only a 5' UTR. In some embodiments, the IL-15 sushi composition comprises only a 3' UTR.

[0697] In some embodiments, the IL-15 sushi RNA comprises a poly-A tail. In some embodiments, the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.

[0698] In some embodiments, the RNA comprises a 5' cap, a 5' UTR, a nucleic acid encoding IL-15 sushi, a 3' UTR, and a poly-A tail, in that order.

[0699] In some embodiments, the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.

[0700] In some embodiments, the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0701] In some embodiments, the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0702] In some embodiments, the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0703] In some embodiments, the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0704] In some embodiments, the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0705] In some embodiments, the IL-15 sushi composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, one or more uridine in the IFN.alpha. RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0706] E. Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

[0707] In some embodiments, the composition comprises a DNA sequence encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) (e.g., SEQ ID NO: 27). In some embodiments, the DNA sequence encoding GM-CSF is provided in SEQ ID NO: 28.

[0708] In some embodiments, the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding GM-CSF. In some embodiments, the RNA sequence is transcribed from SEQ ID NO: 28. The RNA may also be recombinantly produced. In some embodiments, the RNA sequence comprises or consists of SEQ ID NO: 29. In some embodiments, the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 29.

[0709] In some embodiments, one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.). In some embodiments, the GM-CSF RNA comprises an altered nucleotide at the 5' end. In some embodiments, the RNA comprises a 5' cap. Any 5' cap known in the art may be used. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5' to 5' triphosphate linkage including thiophosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3''-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5' cap comprises 7-methylguanylate. In some embodiments, the 5' cap is Cap0 or Cap1. Exemplary cap structures include m7G(5')ppp(5')G, m7,2' O-mG(5')ppsp(5')G, m7G(5')ppp(5')2' O-mG and m7,3' O-mG(5')ppp(5')2' O-mA.

[0710] In some embodiments, the GM-CSF RNA comprises a 5' untranslated region (UTR). In some embodiments, the 5' UTR is upstream of the initiation codon. In some embodiments, the 5' UTR regulates translation of the RNA. In some embodiments, the 5' UTR is a stabilizing sequence. In some embodiments, the 5' UTR increases the half-life of RNA. Any 5' UTR known in the art may be used. In some embodiments, the 5' UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5. In some embodiments, the 5' UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6. In some embodiments, the 5' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.

[0711] In some embodiments, the GM-CSF RNA comprises a 3' UTR. In some embodiments, the 3' UTR follows the translation termination codon. In some embodiments, the 3' UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA. In some embodiments, the 3' UTR RNA sequence is transcribed from SEQ ID NO: 7. In some embodiments, the 3' UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3' UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0712] In some embodiments, the GM-CSF composition comprises both a 5' UTR and a 3' UTR. In some embodiments, the composition comprises only a 5' UTR. In some embodiments, the composition comprises only a 3' UTR.

[0713] In some embodiments, the GM-CSF RNA comprises a poly-A tail. In some embodiments, the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.

[0714] In some embodiments, the GM-CSF RNA comprises a 5' cap, a 5' UTR, nucleotides encoding GM-CSF, a 3' UTR, and a poly-A tail, in that order.

[0715] In some embodiments, the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.

[0716] In some embodiments, the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0717] In some embodiments, the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0718] In some embodiments, the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0719] In some embodiments, the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0720] In some embodiments, the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. The RNA may also be recombinantly produced. In some embodiments, one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m.sup.1.psi.).

[0721] In some embodiments, the GM-CSF composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0722] F. Modifications

[0723] Each of the RNAs and compositions described herein may be modified in any way known to those of skill in the art. In some embodiments, the modifications are "ModA" or "ModB" modified as described herein.

[0724] In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0725] In some embodiments, the modified uridine replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.), or 5-methyl-uridine (m5U).

[0726] In some embodiments, one or more cytosine, adenine or guanine in the RNA is replaced by modified nucleobase(s). In one embodiment, the modified nucleobase replacing cytosine is 5-methylcytosine (m.sup.5C). In another embodiment, the modified nucleobase replacing adenine is N.sup.6-methyladenine (m.sup.6A). In another embodiment, any other modified nucleobase known in the art for reducing the immunogenicity of the molecule can be used.

[0727] The modified nucleoside replacing one or more uridine in the RNA may be any one or more of 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine (mo.sup.5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo.sup.5U), uridine 5-oxyacetic acid methyl ester (mcmo.sup.5U), 5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm.sup.5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U), 5-methoxycarbonylmethyl-uridine (mcm.sup.5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5s.sup.2U), 5-aminomethyl-2-thio-uridine (nm.sup.5s.sup.2U), 5-methylaminomethyl-uridine (mnm.sup.5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm.sup.5s.sup.2U), 5-methylaminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U), 5-carbamoylmethyl-uridine (ncm.sup.5U), 5-carboxymethylaminomethyl-uridine (cmnm.sup.5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm.sup.5s.sup.2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (.tau.m.sup.5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (.tau.m5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m.sup.5s.sup.2U), 1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4.psi.) 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m.sup.3.psi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3.psi.), 5-(isopentenylaminomethyl)uridine (inm.sup.5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5s.sup.2U), .alpha.-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m.sup.5Um), 2'-O-methyl-pseudouridine (.psi.m), 2-thio-2'-O-methyl-uridine (s.sup.2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm.sup.5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm.sup.5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm.sup.5Um), 3,2'-O-dimethyl-uridine (m.sup.3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm.sup.5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0728] G. Combination Compositions

[0729] In some embodiments, the invention comprises a composition comprising more than one RNA as described herein. In some embodiments, the composition comprises two RNAs. In some embodiments, the composition comprises three RNAs. In some embodiments, the composition comprises four RNAs. In some embodiments, the composition comprises five RNAs. In some embodiments, any or all of the RNAs encoding IL-2, IL12sc, IL-15 sushi, GM-CSF, or IFN.alpha. may be replaced by IL-2, IL12sc, IL-15 sushi, GM-CSF, and/or IFN.alpha. polypeptides, e.g., in any of the compositions and formulations comprising these RNAs described herein.

[0730] In some embodiments, the modified or unmodified RNAs encoding IL-2, IL12sc, IL-15 sushi, GM-CSF, and/or IFN.alpha. may be replaced by modified or unmodified polycistronic RNAs encoding two or more polypeptides selected from IL-2, IL12sc, IL15 sushi, GM-CSF and IFN.alpha. polypeptides, e.g., in any of the compositions and formulations comprising these RNAs described herein.

[0731] Any of the combination compositions may further comprise an excipient or diluent. The excipient or diluent may be pharmaceutically acceptable for administration to a subject.

[0732] In some embodiments, a combination composition comprises RNAs with the same modifications. In some embodiments, a combination composition comprises RNAs with different modifications. In some embodiments, a combination composition comprises RNAs with ModA modification. In some embodiments, a combination composition comprises RNAs with ModB modification. In some embodiments, a combination composition comprises RNAs with ModA and ModB modifications.

[0733] In some embodiments, a composition comprising DNA or RNA encoding IL-2 and one or more of a DNA or RNA encoding IL-12sc, IFN.alpha., IL-15 sushi, and GM-CSF is encompassed. In some embodiments, the composition comprises a DNA or RNA encoding IL-2 or codon-optimized IL-2 (SEQ ID NOs: 10-13) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID Nos: 15-18), IFN.alpha. or optimized IFN.alpha. (SEQ ID Nos: 20-23), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein. In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, one or more of the RNAs in the composition further comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail as described herein in the composition section.

[0734] In some embodiments, a composition comprising DNA or RNA encoding IL-12sc and one or more of a DNA or RNA encoding IL-2, IFN.alpha., IL-15 sushi, and GM-CSF is encompassed. In some embodiments, the composition comprises a DNA or RNA encoding IL-12sc or codon-optimized IL-12sc (SEQ ID NOs: 15-18) and one or more of a DNA or RNA encoding IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), IFN.alpha. or optimized IFN.alpha. (SEQ ID NOs: 20-23), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein. In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U).

[0735] In some embodiments, one or more of the RNAs in the composition further comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail, as described herein in the composition section.

[0736] In some embodiments, a composition comprising DNA or RNA encoding IFN.alpha. and one or more of a DNA or RNA encoding IL-2, IL-12sc, IL-15 sushi, and GM-CSF is encompassed. In some embodiments, the composition comprises a DNA or RNA encoding IFN.alpha. or codon-optimized IFN.alpha. (SEQ ID NOs: 20-23) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein. In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, one or more of the RNAs in the composition further comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail as described herein in the composition section.

[0737] In some embodiments, a composition comprising DNA or RNA encoding IL-15 sushi and one or more of a DNA or RNA encoding IL-2, IL-12sc, IFN.alpha., and GM-CSF is encompassed. In some embodiments, the composition comprises a DNA or RNA encoding IL-15 sushi (SEQ ID NOs: 25-26) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IFN.alpha. or optimized IFN.alpha. (SEQ ID NOs: 20-23), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), and GM-CSF (SEQ ID NOs: 28-29), as described herein. In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, one or more of the RNAs in the composition further comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail as described herein in the composition section.

[0738] In some embodiments, a composition comprising DNA or RNA encoding GM-CSF and one or more of a DNA or RNA encoding IL-2, IL-12sc, IFN.alpha., and IL-15 sushi is encompassed. In some embodiments, the composition comprises a DNA or RNA encoding GM-CSF (SEQ ID NOs: 28-29) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IFN.alpha. or optimized IFN.alpha. (SEQ ID NOs: 20-23), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), and IL-15 sushi (SEQ ID NOs: 25-26), as described herein. In some embodiments, one or more uridine in the RNA is replaced by a modified nucleoside as described herein. In some embodiments, the modified nucleoside replacing uridine is pseudouridine (.psi.), N1-methyl-pseudouridine (m.sup.1.psi.) or 5-methyl-uridine (m.sup.5U). In some embodiments, one or more of the RNAs in the composition further comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail as described herein in the composition section.

[0739] In some embodiments, the composition comprises GM-CSF, IL-2, and IL-12sc RNA. In some embodiments, the composition is modified, for example, as ModA or ModB. In some embodiments, the IL-12sc RNA is optimized as shown in SEQ ID NO: 18.

[0740] In some embodiments, the composition comprises GM-CSF, IL-15 sushi, and IL-12sc RNA. In some embodiments, the composition is modified, for example, as ModA or ModB. In some embodiments, the IL-12sc RNA is optimized as shown in SEQ ID NO: 18.

[0741] In some embodiments, the composition comprises GM-CSF, IL-2, IL-12sc, and IFN.alpha. RNA. In some embodiments, the composition is modified, for example, as ModA or ModB. In some embodiments, the IL-12sc RNA and IFN.alpha. RNA is optimized as shown in SEQ ID NOs: 18 and 23, respectively.

[0742] In some embodiments, the composition comprises GM-CSF, IL-15 sushi, IL-12sc, and IFN.alpha. RNA. In some embodiments, the composition is modified, for example, as ModA or ModB. In some embodiments, the IL-12sc RNA and IFN.alpha. RNA is optimized as shown in SEQ ID NOs: 18 and 23, respectively.

[0743] In some embodiments, the composition comprises GM-CSF, IL-15 sushi, IL-12sc, and IFN.alpha. RNA, wherein the RNAs comprise or consist of the nucleotides shown in SEQ ID Nos: 18 (IL-12sc), 23 (IFN.alpha.), 26 (IL-15 sushi), or 29 (GM-CSF). In some embodiments, the composition is modified, for example, as ModA or ModB.

[0744] In some embodiments, combinations of RNA are administered as a 1:1, 1:1:1, or 1:1:1:1 ratio based on equal RNA mass. For example, 20 .mu.g of IL15-sushi, 20 .mu.g of IL-12sc, 20 .mu.g of IFN.alpha.2b and 20 .mu.g GM-CSF. In some embodiments, the ratio is adjusted so that different ratios by mass are administered, for example, 1:10:1:10 ratio (20 .mu.g, 200 .mu.g, 20 .mu.g, 200 .mu.g). Likewise, in some embodiments, for example, a ratio of 1:2:3:4 (20 .mu.g, 40 .mu.g, 60 .mu.g, 80 .mu.g) is used. Alternatively, rather than basing the ratio on the mass of the RNA, the ratio may be based on the molarity of the RNA.

[0745] In some embodiments, a mixture of RNAs is administered with an equal ratio of each RNA of the mixture.

[0746] In some embodiments, a mixture of RNAs is administered with an unequal ratio of each RNA of the mixture. In some embodiments, one or more RNAs are administered at a ratio that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than another RNA in the mixture. In some embodiments, one or more RNAs are administered at a ratio that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times less than another RNA in the mixture.

[0747] In some embodiments, the compositions described herein may be a medical preparation. In some embodiments, the medical preparation comprises a kit, wherein the included RNAs may be in the same or separate vials. In some embodiments, the medical preparation further comprising instructions for use of the composition for treating or preventing a solid tumor.

[0748] In some embodiments, a kit comprising the compositions described herein is provided, wherein the included RNAs may be in the same or separate vials. In some embodiments, the kit further comprising instructions for use of the composition for treating or preventing a solid tumor.

[0749] H. Effect of IFN.alpha. Addition to Modified mRNA Treatment

[0750] RNA can activate the immune system through stimulating various pattern recognition receptors (PRR) leading to production of Type I interferons (like IFN.alpha.). The incorporation of various modified nucleotides, or other alterations like reducing the amount of dsRNA administered, can reduce the immune stimulatory effects of RNA. Unexpectedly, as described in the Examples, inclusion of nucleotide-modified and dsRNA-reduced mRNA encoding interferon alpha improved anti-tumor activity relative to that of mRNA that was not nucleotide modified and dsRNA-reduced. The addition of mRNA encoding interferon alpha restored a portion of the immune stimulatory effects removed by the inclusion of modified nucleotides and the dsRNA purification.

[0751] In some embodiments, RNA encoding IFN (in any form or subtype) is provided, wherein the IFN RNA is altered to have reduced immunogenicity compared to un-altered RNA. In certain embodiments, the administration of this IFN improves the anti-tumor response of non-IFN encoding RNA. In some embodiments, RNA encoding IFN.alpha. improves the anti-tumor response of other RNAs, so long as the other RNAs have been altered to reduce immunogenicity. In one embodiment, the alteration to reduce immunogenicity is a reduction in the amount of dsRNA. In some aspects, the alteration to reduce immunogenicity is the replacement of one or more uridines with a modified nucleoside. In some aspects, the alteration to reduce immunogenicity is both a reduction in the amount of dsRNA and the replacement of one or more uridines with modified nucleoside. In some embodiments, the IFN is IFN.alpha..

[0752] In some embodiments, IFN RNA improves the anti-tumor response of modified RNAs. In some embodiments, IFN RNA improves the anti-tumor response of RNAs comprising modified nucleotides. In some embodiments, IFN RNA improves the anti-tumor response of mRNAs comprising pseudouridine. In some embodiments, IFN RNA improves the anti-tumor response of RNAs with ModB modifications.

[0753] In some embodiments, IFN RNA improves the anti-tumor response of RNAs of IL-2 (SEQ ID NO: 12 or 13), IL-12sc (SEQ ID NO: 17 or 18), IL-15 sushi (SEQ ID NO: 26) or GM-CSF (SEQ ID NO: 29). In some embodiments, the RNAs comprise ModB modifications.

[0754] In some embodiments, the IFN is IFN.alpha..

[0755] In some embodiments, the IFN RNA construct is SEQ ID NO: 22 or 23.

III. Methods and Uses

[0756] Any of the RNAs, compositions, medical preparations and combination compositions described herein may be administered to a subject to treat cancer or a solid tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is an abnormal mass of tissue that does not contain cysts or liquid areas. In some embodiments, the solid tumor may be benign or malignant. In some embodiments, the solid tumor is a pre-cancerous lesion. In some embodiments, the solid tumor occurs in 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.

[0757] In some embodiments, the solid tumor is a sarcoma, carcinoma, or lymphoma. In some embodiments, 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, basal cell carcinoma, squamous cell carcinoma, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor. In some embodiments, the solid tumor is a precancerous lesion such as actinic keratosis.

[0758] In some embodiments, the RNA compositions may be delivered via injection into (e.g., intra-tumorally) or near (peri-tumorally) the tumor. In some embodiments, the RNA compositions may be delivered at or near the site of a tumor removal.

[0759] In some embodiments, the RNA compositions may be delivered via a topical solution, ointment, or cream.

[0760] In some embodiments, the RNA compositions may be delivered via a virus. In some embodiments, the RNA compositions may be delivered by infection with a virus encoding the RNA compositions, such as an oncolytic virus. In some embodiments, the RNA compositions may be delivered by an oncolytic virus.

[0761] In some embodiments, more than one administration is delivered. In some embodiments, a catheter is placed into or near the site of the tumor for multiple administrations. In some embodiments, a catheter is placed at the site of removal of a tumor for multiple administrations.

[0762] In some embodiments, the subject is human. In some embodiments, the subject is a non-human mammal such as a dog, cat, mouse, rat, rabbit, sheep, cattle, horse and pig.

[0763] In some embodiments, RNA compositions are combined with another therapy. In some embodiments, RNA compositions are combined with more than one other therapy. In some embodiments, RNA compositions are combined in a multi-modal therapy.

[0764] In some embodiments, the other therapy is surgery to excise, resect, or debulk the tumor. In some embodiments, therapeutic RNA compositions are administered during a surgery to excise, resect, or debulk the tumor.

[0765] In some embodiments, the other therapy is radiotherapy. In some embodiments, the radiotherapy is external beam radiation therapy or particle beam radiation. In some embodiments, the radiotherapy is brachytherapy involving temporary or permanent implantation of radioactive isotopes directly into the tumor via catheter or large bore needle. In some embodiments, the radioactive isotope is 137Cesium, 192Iridium, or radioactive iodine. In some embodiments, the radiotherapy is radioisotope preparations administered intravenously. In some embodiments, the radioisotope preparations are radioactive iodine (131I) Strontium (89Sr), or Samarium (153Sm).

[0766] In some embodiments, the other therapy is chemotherapy. In some embodiments, the chemotherapy is an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor, or a cytotoxic antibody.

[0767] In some embodiments, the chemotherapy comprises anti-invasion agents (e.g., metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function). In some embodiments, the chemotherapy comprises inhibitors of growth factor function (e.g., platelet derived growth factor and hepatocyte growth factor), growth factor antibodies, or growth factor receptor antibodies, (e.g., anti-erbb2 antibody trastuzumab [Herceptin.TM.] and the anti-erbb1 antibody Cetuximab.TM.). In some embodiments, the chemotherapy is a farnesyl transferase inhibitor. In some embodiments, the chemotherapy is a tyrosine kinase inhibitor such as inhibitors of the epidermal growth factor family (e.g., EGFR family tyrosine kinase inhibitors such as gefitinib (Iressa.TM.), erlotinib (Tarceva.TM.), and Canertinib (CI 1033), or a serine/threonine kinase inhibitor).

[0768] In some embodiments, the chemotherapy comprises antiproliferative/antineoplastic drugs such as antimetabolites (e.g., antifolates like methotrexate, fluoropyrimidines like 5-fluorouracil, tegafur, purine and adenosine analogues, cytosine arabinoside); antitumour antibiotics (e.g., anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (e.g., cisplatin, carboplatin); alkylating agents (e.g., nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (e.g., vinca alkaloids like vincristine, vinblastine, vindesine, vinorelbine, and taxoids like taxol, taxotere); topoisomerase inhibitors (e.g., epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan, camptothecin and also irinotecan); or thymidylate synthase inhibitors (e.g., raltitrexed).

[0769] In some embodiments, the chemotherapy is an antibody-drug conjugate (ADC). In some embodiments, the ADC is an antibody linked to a cytotoxic (anticancer) drug. In some embodiments, the ADC allows targeted delivery of cytotoxic drugs to tumor cells. In some embodiments, the ADC allows preferential deliver of cytotoxic drugs to tumor cells versus normal tissue.

[0770] In some embodiments, the chemotherapy is combination chemotherapy with a combination of different agents. In some embodiments, the combination comprises different agents that have different mechanisms of action and/or different, non-overlapping toxicities.

[0771] In some embodiments, the other therapy is an immune stimulator or immunotherapy, such as, for example, a checkpoint modulator/inhibitor. Checkpoint modulators/inhibitors are well known in the art to prevent the host immune system from attacking itself, and include, for example, CTLA-4, PD1, PDL1, GITR, OX40, LAG-3, and TIM-3. In some embodiments, the immune stimulator, immunotherapy, or checkpoint modulator/inhibitor is a monoclonal antibody. In some embodiments, the monoclonal antibody is an antibody against PD1, PDL1, CTLA-4, LAG3, OX40, CD40, CD40L, 41BB, 41BBL, GITR, CD3, CD28, CD38, or TGFbeta. In some embodiments, the monoclonal antibody is a bispecific antibody. In some embodiments, the immune stimulator is a cell-based immunotherapy. In some embodiments, the immune stimulator is a cytokine or chemokine. In some embodiments, the immune stimulator is a cancer vaccine. As a wide range of immune stimulators would be known to scientists and clinicians skilled in the art, the invention is not limited to a specific combination with a particular immune stimulator.

[0772] In some instances, any of the RNAs, RNA compositions, medical preparations, and RNA combination compositions described herein may be administered in combination with an immune stimulator, immunotherapy, or checkpoint modulator. In some instances, the RNAs, RNA compositions, and RNA combination compositions described herein are administered in combination with an antibody to a subject to treat cancer, including solid tumors. In some embodiments, the antibody is an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination of an anti-PD1 antibody and anti-CTLA4 antibody. In some embodiments, the antibody is a multi-specific antibody such as, for example, a tri-specific or bi-specific antibody.

[0773] In some embodiments, the anti-PD1 antibody is a chimeric, humanized or human antibody. In some embodiments, the anti-PD-1 antibody is isolated and/or recombinant. Examples of anti-PD-1 antibodies are nivolumab, pembrolizumab, cemiplimab, MEDI0608 (formerly AMP-514; see, e.g., WO 2012/145493 and U.S. Pat. No. 9,205,148), PDR001 (see, e.g., WO 2015/112900), PF-06801591 (see, e.g., WO 2016/092419), BGB-A317 (see, e.g., WO 2015/035606).

[0774] In some embodiments, the anti-PD-1 antibody is one of those disclosed in WO 2015/112800 (such as those referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P in Table 1 of the PCT publication, and those referred to as H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 in Table 3 of the PCT publication). The disclosure of WO 2015/112800 is incorporated by reference herein in its entirety. For example, the antibodies disclosed in WO 2015/112800 and related antibodies, including antibodies and antigen-binding fragments having the CDRs, VH and VL sequences, or heavy and light chain sequences disclosed in that PCT publication, as well as antibodies and antigen-binding fragments binding to the same PD-1 epitope as the antibodies disclosed in that PCT publication, can be used in conjunction with the RNA compositions of the present invention to treat and/or prevent cancer.

[0775] In related embodiments, the anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below as SEQ ID NOs: 79 and 80, respectively; the VH and VL sequences in SEQ ID NOs: 87 and 88 (shown in italics), or one or more (e.g., all six) CDRs in SEQ ID NOs: 79 and 80 (shown in bold boxes). In some embodiments, an antibody comprising the following CDRs is encompassed:

TABLE-US-00007 (SEQ ID NO: 81) HCDR1 = GFTFSNFG (SEQ ID NO: 82) HCDR2 = ISGGGRDT (SEQ ID NO: 83) HCDR3 = VKWGNIYFDY (SEQ ID NO: 84) LCDR1 = LSINTF (SEQ ID NO: 85) LCDR2 = AAS (SEQ ID NO: 86) LCDR3 = QQSSNTPFT.

[0776] An exemplary antibody comprising a heavy chain comprising the VH and VL sequences in SEQ ID NOs: 87 and 88 (shown in italics) is the fully human anti-PD-1 antibody known as REGN2810 (cemiplimab).

TABLE-US-00008 Anti-PD-1 Mab heavy chain (SEQ ID NO: 79) ##STR00014## ##STR00015## KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK (SEQ ID NO: 81) HCDR1 = GFTFSNFG (SEQ ID NO: 82) HCDR2 = ISGGGRDT (SEQ ID NO: 83) HCDR3 = VKWGNIYFDY Anti-PD-1 Mab light chain (SEQ ID NO: 80) ##STR00016## ##STR00017## SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 84) LCDR1 = LSINTF (SEQ ID NO: 85) LCDR2 = AAS (SEQ ID NO: 86) LCDR3 = QQ SSNTPFT

[0777] In some embodiments, the RNAs, RNA compositions, and RNA combination compositions may be delivered via injection into the tumor (e.g., intratumorally), near the tumor (peri-tumorally), or near the site of a tumor removal, and the antibody may be delivered in the same manner or systemically, such as, for example, enteral or parenteral, including, via injection, infusion, and implantation. "Administered in combination" includes simultaneous or sequential administration. If sequential, administration can be in any order and at any appropriate time interval known to those of skill in the art.

[0778] In some embodiments, the other therapy is hormonal therapy. In some embodiments, the hormonal therapy is antiestrogen drugs for treatment of breast cancer or anti-androgen drugs for treating prostate cancer. Example agents include antiestrogens (e.g., tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), estrogen receptor down regulators (e.g., fulvestrant), progestogens (e.g., megestrol acetate), aromatase inhibitors (e.g., anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (e.g., flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (e.g., goserelin acetate, luprolide, buserelin), and inhibitors of 5-alpha-reductase (e.g., finasteride).

[0779] In some embodiments, the other therapy is a targeted therapy. In some embodiments, the targeted therapy is a kinase inhibitor. In some embodiments, the targeted therapy is one that inhibits activity of a gene product of a proto-oncogene. In some embodiments, the targeted therapy is an anti-angiogenic agent. In some embodiments, the targeted therapy is one directed to modulate activity of VEGF, BCR-ABL, BRAF, EGFR, c-Met, MEK, ERK, mTOR, or ALK.

[0780] In some embodiments, the other therapy is stem cell transplantation.

[0781] In some embodiments, therapeutic RNA compositions are delivered at the same time as another therapy.

[0782] In some embodiments, therapeutic RNA compositions are delivered before another therapy.

[0783] In some embodiments, therapeutic RNA compositions are delivered after another therapy.

[0784] In some embodiments, therapeutic RNA compositions are delivered directly into the tumor, or near the tumor or the site of tumor removal together with another therapy. In some embodiments, therapeutic RNA compositions are delivered directly into the tumor, or near the tumor or site of tumor removal while another agent is delivered systemically.

IV. Pharmaceutical Formulations

[0785] In some embodiments, any of the DNAs, RNAs, and compositions described herein are pharmaceutical formulations. In some embodiments, the pharmaceutical formulations comprise a diluent, excipient, or other pharmaceutically acceptable carrier. Thus, provided herein are pharmaceutical compositions comprising the DNA, RNA, compositions, or combinations thereof provided herein, and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutically acceptable excipient is an aqueous solution. In certain embodiments, the aqueous solution is a saline solution. As used herein, pharmaceutically acceptable excipients are understood to be sterile. In some embodiments, a pharmaceutical composition is administered in the form of a dosage unit. For example, in certain embodiments, a dosage unit is in the form of a tablet, capsule, implantable device, or a bolus injection.

[0786] In some embodiments, the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions. For example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents. The compositions may also contain additional, compatible, pharmaceutically-inactive materials such as excipients, diluents, and carriers.

[0787] Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

[0788] Lipid moieties may be used to deliver the RNAs provided herein. In one method, the RNA is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In another method, RNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.

[0789] In some embodiments, a pharmaceutical composition provided herein comprises a polyamine compound or a lipid moiety complexed with the DNA or RNA provided herein.

[0790] In some embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0791] Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. In certain embodiments, a pharmaceutical composition provided herein comprises a RNA or combination of RNAs in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to treat or prevent cancer in the subject being treated.

[0792] The following clauses provide numerous embodiments and are non-limiting: [0793] Clause 1. A composition comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein. [0794] Clause 2. The composition of clause 1, wherein the IFN.alpha. protein is an IFN.alpha.2b protein. [0795] Clause 3. The composition of clause 1, 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. [0796] Clause 4. The composition of clause 1, 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. [0797] Clause 5. The composition of clause 1, wherein (i) the RNA encoding an IFN.alpha. 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 IFN.alpha. 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. [0798] Clause 6. The composition of clause 1, 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. [0799] Clause 7. The composition of clause 1, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine, wherein the modified nucleoside is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.), 5-methyl-uridine (m5U), or a combination thereof. [0800] Clause 8. The composition of clause 1, wherein each RNA comprises a modified nucleoside in place of at least one uridine, wherein the modified nucleoside is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.), 5-methyl-uridine (m5U), or a combination thereof [0801] Clause 9. The composition of clause 1, wherein at least one RNA comprises the 5' cap) m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG, or 3'-O-Me-m.sup.7G(5')ppp(5')G, or m.sub.2.sup.7,3'-OGppp(m1.sup.2'-O)ApG, or 3'-O-Me-m.sup.7G(5')ppp(5')G. [0802] Clause 10. The composition of clause 1, wherein at least one RNA comprises 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 and/or 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. [0803] Clause 11. The composition of clause 1, wherein at least one RNA comprises a poly-A tail of at least 100 nucleotides. [0804] Clause 12. The composition of clause 11, wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 78. [0805] Clause 13. The composition of clause 1, wherein one or more RNA comprises: [0806] a 5' cap comprising m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; [0807] 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; [0808] 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 [0809] a poly-A tail comprising at least 100 nucleotides. [0810] Clause 14. The composition of clause 13, wherein the poly-A tail comprises SEQ ID NO: 78. [0811] Clause 15. A pharmaceutical composition comprising the composition of clause 1 and a pharmaceutically acceptable carrier, diluent and/or excipient. [0812] Clause 16. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the composition of clause 1. [0813] Clause 17. 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 IFN.alpha. protein, or a GM-CSF protein, and additional RNA, wherein: [0814] if the first RNA encodes an IL-12sc protein, then the additional RNA encodes an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein; [0815] if the first RNA encodes an IL-15 sushi protein, then the additional RNA encodes an IL-12sc protein, an IFN.alpha. protein, and a GM-CSF protein; [0816] if the first RNA encodes an IFN.alpha. protein, then the additional RNA encodes an IL-15 sushi protein, an IL-12sc protein, and a GM-CSF protein; and [0817] if the first RNA encodes a GM-CSF protein, then the additional RNA encodes an IL-15 sushi protein, an IFN.alpha. protein, and an IL-12sc protein. [0818] Clause 18. The method of clause 17, wherein the first RNA is administered to the subject at the same time as the additional RNA. [0819] Clause 19. The method of clause 17, wherein the RNA is administered intra-tumorally or peri-tumorally. [0820] Clause 20. The method of clause 17, wherein the subject is further treated with an additional therapy comprising (i) surgery to excise, resect, or debulk a tumor, (ii) immunotherapy, (iii) radiotherapy, or (iv) chemotherapy. [0821] Clause 21. The method of clause 17, wherein the subject is further treated with a checkpoint modulator. [0822] Clause 22. The method of clause 21, 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. [0823] Clause 23. The method of clause 21, wherein the RNA is administered intra-tumorally or peri-tumorally via injection, and the checkpoint modulator is administered systemically. [0824] Clause 24. The method of clause 17, wherein the solid tumor is a sarcoma, carcinoma, or lymphoma. [0825] Clause 25. The method of clause 17, 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. [0826] Clause 26. The method of clause 17, 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. [0827] Clause 27. The method of clause 17, wherein treating or reducing the likelihood of the solid tumor comprises reducing the size of a tumor, reducing the likelihood of a reoccurrence of cancer in remission, or reducing the likelihood of cancer metastasis in the subject. [0828] Clause 28. A combination therapy method for treating or reducing the likelihood of a solid tumor, comprising administering to a subject in need thereof RNAs and a further therapy, wherein the RNAs encode an IL-12sc protein, an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein, and the further therapy comprises immunotherapy, chemotherapy, or a checkpoint modulator. [0829] Clause 29. The combination therapy method of clause 28, wherein the further therapy comprises an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody. [0830] Clause 30. An isolated nucleic acid comprising a sequence encoding an IL-12sc protein, wherein: [0831] the sequence encoding the IL-12sc protein comprises a) contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 78% identity to nucleotides 1-984 of SEQ ID NO: 16 or 18, b) contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16 or 18, and c) nucleotides encoding a linker between the nucleotides of a) and b). [0832] Clause 31. The nucleic acid of clause 30, which is a DNA. [0833] Clause 32. The nucleic acid of clause 30, which is an RNA. [0834] Clause 33. An isolated nucleic acid comprising a sequence encoding an IFN.alpha. protein, wherein: [0835] the sequence encoding the IFN.alpha. protein has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21 or 23. [0836] Clause 34. The nucleic acid of clause 33, which is a DNA. [0837] Clause 35. The nucleic acid of clause 33, which is an RNA. [0838] Clause 36. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the RNA of clause 34, wherein: the subject is further treated with additional RNA encoding an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein. [0839] Clause 37. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the RNA of clause 35, wherein: the subject is further treated with additional RNA encoding an IL-15 sushi protein, an IL-12sc protein, and a GM-CSF protein. [0840] Clause 38. A method of producing an RNA encoding IL-12sc, comprising contacting an expression construct comprising the nucleic acid of clause 32 operably linked to a promoter with an RNA polymerase under conditions permissive for transcription. [0841] Clause 39. A method of producing an RNA encoding IFN.alpha., comprising contacting an expression construct comprising the nucleic acid of clause 35 operably linked to a promoter with an RNA polymerase under conditions permissive for transcription. [0842] Clause 40. A kit comprising the composition of clause 1. [0843] Clause 41. A kit comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein, wherein the RNAs are not in the same container. [0844] Clause 42. The kit of clause 41, wherein each RNA is in a separate container. [0845] Clause 43. The kit of any one of clause 40-42, further comprising instructions for use of the composition or RNAs for treating or reducing the likelihood of a solid tumor.

[0846] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about," to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0847] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

EXAMPLES

[0848] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

Example 1--Materials and Methods

[0849] B16F10 Tumor Model:

[0850] Female C57BL/6J mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and housed on 12 hours light/dark cycle at 22.degree. C..+-.2.degree. C. with a relative humidity of 55%.+-.15%. B16F10 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, Va. USA) (Cat No. CRL-6475) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life technologies, Cat No. 11995) supplemented with 10% heat inactivated Fetal Bovine Serum (HI FBS) (Life technologies, Cat No. 10082-147) in 5% CO2 at 37.degree. C. The cells were harvested using 0.25% Trypsin-EDTA (Life technologies, Cat No. 25200-056), resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Life technologies, Cat No. 14190-144), and 0.5.times.10{circumflex over ( )}6 cells/200 .mu.l per mouse subcutaneously (SC) implanted into the right flank of female C57BL/6J mice. For dual flank tumor models on day 0, 0.5.times.10{circumflex over ( )}6 B16F10 cells/200 .mu.l per mouse were subcutaneously (SC) implanted into the right flank and 0.25.times.10{circumflex over ( )}6 B16F10 cells/200 .mu.l per mouse were SC implanted into the left flank. To test whether local administration of cytokine mRNA can have distant effects (see FIG. 19), female C57BL/6JOlaHsd mice (Envigo, Rossdorf, Germany), 6-8 weeks of age, with a weight between 17 and 24 g, were acclimated for at least six days prior to study enrollment. Mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and housed on 12 hours light/dark cycle at 22.degree. C..+-.2.degree. C. with a relative humidity of 55%.+-.10%. B16F10 cells were obtained from the American Type Culture Collection (ATCC.RTM. CRL-6475.TM.) and cultured in DMEM, high glucose, GlutaMAX.TM. (Life technologies, Cat No. 31966047) supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom, Cat No. S 0115) in 7.5% CO2 at 37.degree. C. The cells were harvested using StemPro.RTM. Accutase.RTM. Cell Dissociation Reagent (Life technologies, Cat No. A1110501), re-suspended in Dulbecco's phosphate-buffered saline (DPBS) (Life technologies, Cat No. 14190-169), and 0.3.times.10{circumflex over ( )}6 cells/100 .mu.l per mouse subcutaneously (SC) were implanted into the right shaven flank of female C57BL/6J mice. For seeding of distant lung tumors, B16F10_luc-gfp cells were used. These cells were derived from B16F10 by stable transfection with a plasmid coding for Luciferase and GFP. B16F10_luc-gfp cells were cultured with the same conditions as the B16F10; only 0.5 .mu.g/mL Puromycin was added to the culture medium. One day after SC implantation 0.3.times.10{circumflex over ( )}6 cells/200 .mu.l of B16F10_luc-gfp cells were injected per mouse intravenously (IV) into the tail vein. Intratumoral mRNA injections were initiated 10-14 days after SC inoculation of the tumors. Tumor growth was assessed by caliper measurements every 2-3 days and is expressed as the product of the perpendicular diameters using the following formula: a2*b/2, with a<b. Engraftment of luciferase-positive tumor cells in the lung was analyzed by in vivo bioluminescence imaging using the Xenogen IVIS Spectrum imaging system (Caliper Life Sciences). An aqueous solution of L-luciferin (2500, 1.6 mg; BD Biosciences) was injected intraperitoneally. Emitted photons from live animals quantified 10 min later with an exposure time of 1 min. Regions of interest (ROI) were quantified as average radiance (photons s.sup.-1 cm.sup.-2'sr.sup.-1, represented by as color-scaled images superimposed on grayscale photos of mice using the Living Image software from Caliper Life Sciences). For absolute quantification, the bioluminescence signal was blotted as total flux (photons s.sup.-1). For studies performed in FIGS. 20C-G and 21E-I the above described model of C57BL/6JOlaHsd mice (Envigo, Rossdorf, Germany) without seeding of lung metastasis was used as well.

[0851] CT26 Tumor Model:

[0852] For studies performed in FIGS. 2, 3, 7D-F, 8, 9, 12D and 21, female Balb/c Rj mice (Janvier, Genest-St.-Isle, France), 6-8 weeks of age, with a weight between 17 and 24 g, were acclimated for at least six days prior to study enrollment. Mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and housed on 12 hours light/dark cycle at 22.degree. C..+-.2.degree. C. with a relative humidity of 55%.+-.10%. CT26 cells were obtained from the (ATCC.RTM. CRL-2638.TM.) and cultured in Roswell Park Memorial Institute medium (RPMI) 1640 Medium, GlutaMAX.TM. (Life technologies, Cat No. 61870-044) supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom, Cat No. S 0115) in 5% CO2 at 37.degree. C. The cells were harvested using StemPro.RTM. Accutase.RTM. Cell Dissociation Reagent (Life technologies, Cat No. A1110501), resuspended in DPBS (Life technologies, Cat No. 14190-169), and 0.5.times.10{circumflex over ( )}6 cells/100 .mu.l per mouse SC implanted into the right shaven flank of female Balb/c Rj mice. Intratumoral RNA injections were initiated 13-19 days after inoculation of the tumors. Tumor growth was assessed by caliper measurements every 2-3 days and is expressed as the product of the perpendicular diameters using the following formula: a2*b/2 where b is the longer of the two diameters (a<b).

[0853] For studies performed in FIG. 6, and 7A-C female BALB/c mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and housed on 12 hours light/dark cycle at a temperature (22.degree. C..+-.2.degree. C.), relative humidity (55%.+-.15%). CT26 cells were obtained from the ATCC (Manassas, Va. USA) (Cat No. CRL-2638) and cultured in RPMI-1640 (Life technologies, Cat No. 11875-093) supplemented with 10% HI FBS (Life technologies, Cat No. 10082-147) in 5% CO2 at 37.degree. C. The cells were harvested using 0.25% Trypsin-EDTA (Life technologies, Cat No. 25200-056), re-suspended in DPBS (Life technologies, Cat No. 14190-144), and 0.5.times.10{circumflex over ( )}6 cells/200 .mu.l per mouse SC implanted into the right flank of BALB/c female mice.

[0854] In CT26 tumor model in addition to tumor growth gp70-reactive CD8+ T-cells were measured in blood where indicated. Blood samples were taken using EDTA-coated tubes. 100 .mu.L of blood was transferred to FACS tubes and antibody mixture was added containing T-Select H-2Ld MuLV gp70 Tetramer-SPSYVYHQF-APC (MBL (TS-M-521-2), 4 .mu.L for 100 .mu.L blood), anti-CD8a FITC (life technologies (MCD801), 1 .mu.L for 100 blood)) and anti-CD45 V500 (BD (561487), 1 .mu.L for 100 .mu.L blood)). After 20 min incubation at room temperature Blood Lysis Buffer (BD (349202), 300 .mu.L per tube) was added and incubated for further 6 min. Then samples were washed twice with PBS-EDTA buffer. FACS samples were analyzed on a FACS Canto II flow cytometer.

[0855] MC38 Tumor Model:

[0856] Female C57BL/6J mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and housed on 12 hours light/dark cycle at 22.degree. C..+-.2.degree. C. with a relative humidity of 55%.+-.15%. MC38 cells were generous gifts from Dr S. A. Rosenberg (National Institute of Health, Bethesda, Md., USA). The cell line was cultured in RPMI-1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No. 100082) in 5% CO2 at 37.degree. C. The cells were harvested, re-suspended in DPBS (Gibco, Cat No. 14190), and 1.times.10{circumflex over ( )}6 cells/200 .mu.l per mouse SC implanted into the right flank of female C57BL/6J mice.

[0857] A375 Tumor Model:

[0858] Female severe combined immune deficiency (SCID) mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and housed on 12 hours light/dark cycle at 22.degree. C..+-.2.degree. C. with a relative humidity of 55%.+-.15%. A375 cells were obtained from the ATCC (Manassas, Va. USA) (Cat No. CRL-1619). The cell line was cultured in DMEM (Life technologies, Cat No. 11995) supplemented with 10% HI FBS (Life technologies, Cat No. 10082-147) in 5% CO2 at 37.degree. C. The cells were harvested using 0.25% Trypsin-EDTA (Life technologies, Cat No. 25200-056), re-suspended in DPBS (Life technologies, Cat No. 14190-144), and 3.0.times.10.sup.6/100 .mu.l PBS were mixed with 100 ul BD Matrigel Matrix (BD, Cat No. 354234) and implanted SC into the right flank of female SCID mice.

[0859] KM12 (CRC) Xenograft Model: Female NOD.CB17-Prkdcscid/SCID mice (Jackson Laboratory, Bar Harbor, Me.), 10-weeks-old and weighing between 17.3 g and 21.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22.+-.2.degree. C.) with a relative humidity (55.+-.15%). KM-12 cells were obtained from the American National Cancer Institute (NCI) (Cat No. 507345). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No. 10082), and incubated at 37.degree. C. with 5% CO2. The cells were harvested using 0.25% Trypsin-EDTA (Gibco, Cat No. 25200), re-suspended in DPBS (Gibco, Cat No. 14190), and for each mouse 5.0.times.106 cells in 200 .mu.l DPBS with 50% matrigel (BD, Cat No. 356234) were SC implanted into the right flank of female SCID mice.

[0860] RPMI8226 (Myeloma) Xenograft Model: Female NSG mice (Jackson Laboratory, Bar Harbor, Me.), 12-weeks-old and weighing between 19.8 g and 26.6 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22.+-.2.degree. C.) with a relative humidity (55.+-.15%). RPMI8226 cells were obtained from the ATCC (Cat No. CCL-155). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No. 10082), and incubated at 37.degree. C. with 5% CO2. The cells were harvested using 0.25% Trypsin-EDTA (Gibco, Cat No. 25200), re-suspended in DPBS (Gibco, Cat No. 14190), and for each mouse 5.0.times.106 cells in 200 .mu.l DPBS with 50% matrigel (BD, Cat No. 356234) were SC implanted into the right flank of female NSG mice.

[0861] NCI-N87 (Gastric) Xenograft Model: Female NOD.CB17-Prkdcscid/SCID (Jackson Laboratory, Bar Harbor, Me.), 11-weeks-old and weighing between 18.3 and 22.7 g were acclimated for at least three days before the study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22.+-.2.degree. C.) with a relative humidity (55.+-.15%). NCI-N87 cells were obtained from the ATCC (Cat No. CRL-5822). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No. 10082), and incubated at 37.degree. C. with 5% CO2. The cells were harvested using 0.25% Trypsin-EDTA (Gibco, Cat No. 25200), re-suspended in DPBS (Gibco, Cat No. 14190), and for each mouse 3.0.times.106 cells in 200 .mu.l DPBS with 50% matrigel (BD, Cat No. 356234) were SC implanted into the right flank of female SCID mice.

[0862] NCI-H1975 (NSCLC) Xenograft Model: Female NSG mice (Jackson Laboratory, Bar Harbor, Me.), 10-weeks-old and weighed between 18.8 g and 26.0 g were allowed to acclimate for at least three days before study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22.+-.2.degree. C.) with a relative humidity (55.+-.15%). NCI-H1975 cells were obtained from the ATCC (Cat No. CRL-5908) and cultured in RPMI medium 1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No. 10082), and incubated at 37.degree. C. with 5% CO2. The cells were harvested using 0.25% Trypsin-EDTA (Gibco, Cat No. 25200), re-suspended in DPBS (Gibco, Cat No. 14190), and for each mouse 5.0.times.106 cells in 200 .mu.l DPBS with 50% matrigel (BD, Cat No. 356234) were SC implanted into the right flank of female NSG mice.

[0863] Tumor Re-Challenge:

[0864] Female C57BL/6J mice were implanted with B16F10 cells as described above. Mice were treated with 4 intratumoral injections (80 .mu.g mRNA/20 .mu.g per target) on days 11, 13, 15, and 17 with ModB cytokine mRNA mixture (IL-15sushi, IL-12sc, GM-CSF, IFN.alpha.). After cytokine mRNA treatment 8 mice were tumor free. Four weeks after the last cytokine mRNA treatment tumor free mice were re-challenged with 0.5.times.10{circumflex over ( )}6 B16F10 cells/200 .mu.l per mouse by SC injection and tumor growth was monitored.

[0865] Tumor Monitoring:

[0866] Tumors were measured with a caliper twice weekly until final sacrifice. When a tumor size reached approximately 2000 mm.sup.3 or there are animal health issues (20% area of a tumor ulcerated), animals were euthanized. Tumor regression was defined as i) tumor volume<20 mm3 at the end of the study or ii) T.sup.F/T.sup.0<1, where the T.sup.F equals the final tumor volume and T.sup.0 equals tumor volume on the day of the first intratumoral mRNA injection.

[0867] mRNA Modification a (ModA):

[0868] Synthetic DNA fragments coding for the gene of interest were cloned into a common starting vector, comprising a 5'-UTR (corresponding in some cases to SEQ ID NO: 1), a 3' UTR consisting of two elements called F and I (corresponding in some cases to SEQ ID NO: 7), and a poly(A)-tail of 110 nucleotides in total (A30-linker-A70 structure; corresponding in some cases to SEQ ID NO: 78). Linearization of plasmid DNA was performed downstream of the poly(dA:dT) with a classIIS restriction enzyme to generate a template with no additional nucleotide beyond the poly(dA:dT) (see Holtkamp et al., Blood 108(13):4009-172006 (2006)). Linearized plasmid DNA was subjected to in vitro transcription with T7 RNA polymerase (Thermo Fisher) as previously described (see Grudzien-Nogalska E et al., Methods Mol Biol. 969:55-72 (2013)) in the presence of 7.5 mM ATP, CTP, UTP, GTP and 6 mM D1, a beta-S-antireverse cap analogue (beta-S-ARCA, Cap0) (see Kuhn A N et al, Gene Ther. 17(8):961-71 (2010)). RNA was purified using magnetic particles (see Berensmeier S. Appl Microbiol Biotechnol. 73(3):495-504 (2006)), and RNA concentration and quality were assessed spectrophotometry and analyzed by capillary gel electrophoresis systems, respectively.

[0869] mRNA Modification B (ModB):

[0870] Synthetic DNA fragments coding for the gene of interest were cloned into a common starting vector, comprising a 5'-UTR (corresponding in some cases to the Tobacco Etch Viral leader sequences TEV, SEQ ID NO: 3), a 3' UTR consisting of two elements called F and I (corresponding is some cases to SEQ ID NO: 7), and a poly(A)-tail of 110 nucleotides in total (A30-Linker-A70 structure). Upon linearization of plasmid DNA as described above, in vitro transcription with T7 RNA polymerase (Thermo Fisher) was performed. This was carried out as described for ModA, but no cap structure was added to the reaction and UTP was substituted for N1-methyl-pseudouridinetriphosphate. RNA was then purified using magnetic particles (Berensmeier 2006), and subsequently Cap1 structure was enzymatically introduced using a commercially available system based on the Vaccinia virus capping enzyme (NEB) and addition of mRNA Cap 2'-O-methyltransferase (NEB). Afterwards, the RNA was subjected to a further purification procedure by Cellulose-based chromatography to remove double-stranded RNA impurities (see Day P R et al, Phytopathology 67:1393 (1977); Morris T J et al., Phytopathology 69:854-858 (1979); and Castillo A et al., Virol J. 8:38 (2011)). RNA concentration and quality were assessed spectrophotometry and analyzed by capillary gel electrophoresis systems, respectively. Presence of dsRNA was assessed in a Northwestern dot-blot assay using dsRNA-specific J2 mAb (English & Scientific Consulting) as described in Kariko et al. Nucleic Acids Res. 39(21):e142 (2011).

[0871] mRNA Codon Optimization:

[0872] The coding sequence of a protein may influence the efficiency as well as the accuracy of protein translation (see Bossi L et al., Nature. 286(5769):123-7 (1980) and Irwin et al., J Biol Chem. 270(39):22801-6 (1995)).

[0873] Therefore, different codon variants of each target were designed and tested. The design of the different codon variants for each target utilized publicly available software from Life Technologies GmbH GeneArt.RTM. (Regensburg, Germany) (see Raab D et al., Syst Synth Biol. 4(3):215-25 (2010)) and Eurofins MWG Operon (Ebersberg, Germany). In addition, codon optimization was performed manually editing each codon separately. A GC-content comparable to the wild type sequence was maintained during the optimization process.

[0874] Evaluation and selection of constructs was determined by in vitro expression performed using: (i) mRNA lipofection of HEK293T/17 cells and (ii) mRNA electroporation K562 cells.

[0875] Forty thousand (40,000) HEK293T/17 cells (ATCC.RTM. CRL-11268.TM.) were seeded in flat bottom 96-well plates (VWR International, Cat No. 734-1794) in DMEM, high glucose, GlutaMAX.TM. (Life technologies, Cat No. 31966047) containing 0.5% FBS (Biochrom, Cat No. S 0115). Seeded cells in 96-well plates were incubated at 37.degree. C., 7.5% CO2 for 16-18 hours. Adherent HEK293T/17 cells were transfected with RNA using Lipofectamine Messenger MAX Reagent (Invitrogen, Cat No. LMRNA) according to the manufacturer's protocol by adding 1.2 .mu.l of the transfection reagent to 20 .mu.l of OptiMEM (Thermo Fisher, Cat No. 31985070) in an RNAse-free 1.5 ml Safe-Lock tube biopur (Eppendorf, Germany, Cat No. 0030121589); in a second tube the indicated RNA was added to 20 .mu.l of OptiMEM. After 10 minutes of incubation the tube containing the RNA was diluted into the tube containing the Lipofectamine Messenger MAX and incubated an additional 5 minutes prior to adding 10 .mu.l of the RNA-lipid-complex drop wise to one well of the 96-well plate containing the HEK293T/17 cell layer in 100 .mu.l medium. The 10 .mu.l RNA-lipid-complex contained 5 ng, 25 ng and 100 ng of target RNA respectively. The plates were placed into the incubator for 3 h before an additional 140 .mu.l of fresh medium (DMEM+0.5% FBS) was added. The transfected cells were incubated for 15-18 hours and the supernatants were collected and analyzed for protein content by ELISA as described herein.

[0876] K562, a human cell line derived from chronic myeloid leukemia (ATCC.RTM. CCL-243.TM.) was cultivated in RPMI 1640 Medium, GlutaMAX.TM. (Life technologies, Cat No. 61870-044) supplemented with 5% FBS. K562 were electroporated in a 96-well plate system as follows. Cells were washed once in X-VIVO15 medium (Lonza, Cat No. BE02-060Q) and suspended to a final concentration of two hundred fifty thousand (250,000) cells/150 .mu.l in X-VIVO15. A 150 .mu.l of cell suspension was added per well of a 96-well plate containing 5 ng, 25 ng, or 100 ng of RNA. Cells and RNA were mixed and electroporation was performed in a 96 well Gene Pulser MX cell electroporation system from Biorad (250 V, 1.times.30 ms pulse). Immediately following electroporation, cells were transferred into a new culture plate with fresh medium without antibiotics and rested for 1 hour in the incubator at 37.degree. C. The medium was exchanged for fresh RPMI 1640 GlutaMAX supplemented with 0.5% FCS and incubated 15-18 hours. Supernatants were harvested and analyzed for protein content by ELISA as described herein.

[0877] Protein concentrations were determined by ELISAs specific for the RNA encoded cytokine according to the manufacturer's protocol. (i) Human IL-15 sushi/IL-15 sushi R alpha Complex DuoSet ELISA (ii) Mouse IL-12sc Duo Set Development System (DY419-05) (iii) Mouse GM-CSF DuoSet ELISA Development Systems (DY415), all obtained from RnD systems, and mouse IFN.alpha. ELISA Kit (TCM) (PBL assay science, 42120-2).

[0878] For each mRNA target, the protein expression was evaluated for the wt-sequence and the different codon-optimized variants. Both data sets from lipofection of HEK293T/17 and electroporation of K562 each tested with the three different amounts of modified RNA were considered for selection of the protein coding sequence. A codon-optimized sequence was selected if an at least 1.5-fold increase of protein expression compared to WT sequence was measured. If this was not the case, the WT sequence was selected. For all constructs Earl-restriction sequences were eliminated by mutating the DNA recognition sequence (5'-CTCTTC-3'), while preserving the WT amino acid.

[0879] Cell Lines:

[0880] HEK293 (ATCC CRL-1573) cell line and human melanoma cell lines, A101D (ATCC CRL-7798), A375 (ATCC CRL-1619), A2058 (ATCC CRL-11147), and Hs294T (ATCC HTB-140), were obtained from ATCC and cultured in DMEM (ThermoFisher Scientific, Cat 11885-084) supplemented with 10% FBS HI FBS Life Technologies, Cat. 10082) in a humidified atmosphere of 5% CO2 at 37.degree. C.

[0881] mRNA Transfection:

[0882] Cells were transfected using Lipofectamine MessengerMAX Reagent (Invitrogen, Cat # LMRNA001) according to the manufacturer protocol. Briefly, for each well 0.3 .mu.l of the transfection reagent was diluted with 5 .mu.l of the Opti-MEM media (Life Technologies, Cat. 31985062) and incubated for 10 min at room temperature; mRNA mixtures were diluted with Opti-MEM media (5 .mu.l per well) and mixed with diluted MessengerMax reagent, incubated for 5 min at room temperature and aliquoted to the 96-well plate. Cells were diluted in complete growth media and 40,000 cells per well were added to the transfection mixtures. Cells were incubated for 24 hours at 37.degree. C. in a CO2 incubator then media was collected and cytokine concentration was determined by Meso Scale Discovery (MSD) assay.

[0883] Meso Scale Discovery Assay:

[0884] Cytokine concentration was determine using MSD assays: Proinflammatory Panel 1 (human) MSD kit (catalog N05049A-1) for IL12p70, Cytokine Panel 1 (human) MSD kit (catalog N05050A-1) for GM-CSF and IL-15 sushi, and Human IFN-.alpha. 2a Ultra-sensitive Kit (catalog N05050A-1) for IFN.alpha.. Data were analyzed using MSD Discovery Workbench V. 4.0.12 software and GraphPad Prism V.6.00 software.

[0885] PBMC Isolation and Treatment:

[0886] Peripheral Blood Mononuclear Cells (PBMCs) were isolated by density gradient media (Ficoll-Paque) from a leukopak (Research Blood Components). 600,000 PBMCs were added per well of a 96-well plate. Cells were treated with a cytokine mixture for 24 hrs and IFN.gamma. production was measured in the cell culture media using Human IFN.gamma. 384-Tissue Culture MSD Assay.

[0887] Measuring CD8 Response:

[0888] B16F10 tumor bearing mice received a single intratumoral injection of Immuno mRNA (IFN.alpha., IL-15 sushi, GM-CSF, and IL-12sc, ModB) or control luciferase mRNA (Placebo). Seven days after intratumoral mRNA injection tumors were excised, processed for immunofluorescence and stained with an antibody for CD8 (gray). As shown in FIG. 25, CD8 cells were present after cytokine mRNA intratumoral injection.

[0889] Preparation of mRNA for In Vivo Studies:

[0890] The respective mRNA mixtures were prepared for in vivo studies by mixing equal quantities (micrograms) of mRNA in water at 2.times. the intended dose. The mRNA mixture was frozen at -80 C until the day of intratumoral injection. On the day of injection, mRNA was thawed and mixed with an equal volume of 2.times. sterile Ringer's solution. The resulting 1.times.mRNA/Ringer solution was used for intratumoral injection.

[0891] Gene Expression Analysis:

[0892] A375 tumors were homogenized in the RLT Buffer (Qiagen) using Precellys 24 homogenizer (Bertin Instruments). Total RNA was isolated with the RNeasy-96 Kit (Qiagen), following the spin protocol with the DNase treatment. RNA was eluted with nuclease-free water and quantified by ultraviolet absorbance using a NanoDrop 8000 (Thermo Scientific). cDNA synthesis was performed with the High Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer's recommendations. Real-time PCR were performed on a ViiA.TM.7 (Applied Biosystems) according to standard protocol. Amplification was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems) and predesigned Taqman Assays (Applied Biosystems). Gene expression was normalized to the endogenous control GAPDH. Comparative ddCT method was used to evaluate gene expression.

TABLE-US-00009 TABLE 3 Gene Assay ID Lot Number Fluorescent dye ISG15 Hs01921425_s1 1548867 FAM ISG54 Hs01922738_s1 1532771 FAM Mx1 Hs00895608 m1 1539223 FAM hGAPDH 4326317E 1311049 VIC

Example 2--Combinations of Three mRNAs Reduce Tumor Volume In Vivo

[0893] A mixture of modified mRNAs encoding GM-CSF, IL-2, and IL-12sc was injected into B16F10 tumor bearing mice and tumor growth was monitored to day 41. As shown in FIG. 1, intratumoral injection of a combination of three mRNAs encoding GM-CSF, IL-2, and IL-12sc having ModA (SEQ ID NOs: 32, 38, and 56; FIG. 1A), or a combination of three mRNAs encoding GM-CSF, IL-2, and IL-12sc having ModB (SEQ ID NOs: 35, 41, and 59; FIG. 1B) induced regression in 6 out of the 10 mice, while mice treated with a control mRNA encoding luciferase (ModA) displayed tumor regression in 1 of 10 animals (FIG. 1C). These data were confirmed in a repeat study of similar design (FIG. 1D-1G). In the repeat experiment, a mixture of cytokine mRNA (GM-CSF, IL-2, IL-12sc) with ModA or ModB lead to tumor regression in 5 of 9 mice, while treatment with control mRNA in ModA and ModB displayed 2 of 9 and 0 of 9 tumor regressions, respectively.

[0894] The effects of cytokine mRNA treatment were evaluated in CT26 tumors. Mice with established CT26 tumors were injected with a cytokine mRNA mixture encoding GM-CSF, IL-2, and IL-12sc in ModA and ModB formats, respectively. Two control groups were included: i) mRNA Ringer's diluent and ii) ModA mRNA encoding firefly luciferase. A total of 6 intratumoral injections were administered on days 19, 21, 24, 26, 28 and 31. As shown in FIG. 2, both GM-CSF, IL-2, and IL-12sc mRNA ModA (SEQ ID NOs: 56, 32, and 38; FIG. 2A) and ModB (SEQ ID NOs: 59, 35, and 41; FIG. 2B) resulted in tumor regression in 5 and 6 out of 8 mice, respectively, while no tumors treated with control mRNA in ModA (FIG. 2C) or Ringer's solution (FIG. 2D) displayed tumor regression.

[0895] The cytokine mRNA mixture encoding IL-15 sushi, GM-CSF and IL-12sc (ModB; SEQ ID NOs: 53, 59, and 41) and IL-2, GM-CSF and IL-12sc (ModB; SEQ ID NOs: 35, 59, and 41) were evaluated for anti-tumor activity in the CT26 tumor model. Tumors received intratumoral mRNA injections on days 13, 15, 18, 20 and 22 after tumor inoculation. As shown in FIG. 3, intratumoral injection of either the IL-2 mixture (FIG. 3A) or IL-15 sushi mixture (FIG. 3B) resulted in tumor regression in 5 out of 10 or 11 tumor-bearing animals, respectively (FIGS. 3A and B), whereas in the control group injected with luciferase mRNA (ModB) no tumor regression was observed (FIG. 3C).

[0896] Anti-tumor activity of the mRNA mixture of IL-2, IL-12sc, and GM-CSF (FIG. 4A-ModA [SEQ ID NOs: 32, 38, and 56], 4B-ModB [SEQ ID NOs: 35, 41, and 59]), or IL-15 sushi, IL-12sc, and GM-CSF or (FIG. 4C-ModA [SEQ ID NOs: 50, 38, and 56], 4D-ModB [SEQ ID NOs: 53, 41, and 59) was further evaluated in the B16F10 tumor model. Mice with B16F10 tumors were injected intratumorally with mRNA on days 11, 13, 15, and 17. Treatment of B16F10 tumors with the cytokine mRNA mixture that encoded IL-15 sushi, IL-12sc, GM-CSF resulted in tumor regression in 3 and 4 of 8 mice in ModA and ModB, respectively. Tumors treated with an mRNA mixture of IL-2, IL-12sc, and GM-CSF (ModA or ModB) had tumor regression in 4 out of 8 mice, while no tumor regression was noted for mice treated with ModA or ModB mRNA encoding luciferase (FIG. 4E-ModA, FIG. 4F-ModB).

Example 3--Combinations of Four mRNAs Reduce Tumor Volume In Vivo

[0897] We next tested the effect of adding a fourth mRNA to the cytokine mRNA mixture. B16F10 tumor bearing mice received four intratumoral injections of ModB cytokine mRNA mixture encoding: i) GM-CSF, IL-2, IL-12sc (SEQ ID NOs: 59, 35, and 41; FIG. 5A), ii) GM-CSF, IL-15 sushi, IL-12sc (SEQ ID NOs: 59, 53, and 41; FIG. 5B) and iii) GM-CSF, IL-15 sushi, IL-12sc, IFN.alpha. (SEQ ID NOs: 59, 53, 41, and 47; FIG. 5C), and tumor growth was monitored to day 45. Each of the cytokine mRNA mixtures had an anti-tumor effect with 4 out of 8 tumors regressing following intratumoral injection of cytokine mRNA mixtures of GM-CSF, IL-2, and IL-12sc or GM-CSF, IL-15 sushi, and IL-12sc, and 7 out of 8 tumors regressed upon treatment with GM-CSF, IL-15 sushi, IL-12sc, and IFN.alpha.. Mice treated with control mRNA (ModB) exhibited no tumor regression (FIG. 5D).

[0898] The anti-tumor activity of GM-CSF, IL-2, IL-12sc, and IFN.alpha. was examined in three different murine in vivo tumor models, CT26, B16F10 and MC38. Tumor bearing mice received 4-6 intratumoral injections of ModB cytokine mRNA encoding IL-2, IL-12sc, GM-CSF and IFN.alpha. (SEQ ID NOs: 35, 41, 59, and 47) or a control ModB mRNA encoding firefly luciferase. Anti-tumor activity was assessed in each tumor model. Mice treated with this combination of cytokine mRNA had 4/8, 7/8 and 5/5 regressing tumors in the CT26 (FIG. 6A), B16F10 (FIG. 6C) and MC38 (FIG. 6E) models, respectively. No tumor regression was observed in the comparator tumor bearing mice treated with luciferase mRNA (FIGS. 6B [CT26], 6D [B16F10], and 6F [MC38]).

[0899] The anti-tumor activity of a four-component cytokine mRNA mixture encoding each of IL-2, IL-12sc, GM-CSF and IFN.alpha. (ModB, SEQ ID NOs: 35, 41, 59, and 47) or IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. (ModB, SEQ ID NOs: 53, 41, 59, and 47) was evaluated in the CT26 tumor model. Tumor bearing mice were treated with 6 intratumoral injections and tumor growth was monitored. Treatment with both IL-2, IL-12sc, GM-CSF and IFN.alpha. (FIG. 7A) and IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. (FIG. 7B) effectively induced tumor regression in 4/8 and 8/8 mice, respectively, while no tumor regressions were observed for mice treated with the control mRNA (FIG. 7C). These data were confirmed in a repeat study of similar design (FIG. 7D-F).

[0900] A study in CT26 tumor model was conducted in which individual components were systematically removed from the mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. (ModB, SEQ ID NOs: 53, 41, 59, and 47). CT26 tumors were injected with cytokine mRNA on days 12, 15, 19 and 22 after inoculation. Tumors treated with four injections of mRNA encoding IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. induced regression in all 10 treated tumors (FIG. 8A). Tumors treated with the ModB mRNA mixtures of i) IL-15 sushi, IL-12sc and IFN.alpha., ii) IL-15 sushi, GM-CSF and IFN.alpha., iii) IL-12sc, GM-CSF and IFN.alpha., iv) IL-15 sushi, IL-12sc, and GM-CSF resulted in regression of 8, 6, 8, 7 out of 10 tumors, respectively (FIG. 8B-E). No tumors treated with control mRNA (ModB) displayed tumor regression (FIG. 8F). To analyze tumor growth kinetics, mean tumor volumes were calculated for each treatment group up to day 33 (FIG. 8G). The smallest mean tumor volume was observed for mice treated with the mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha., while the largest mean tumor volume was observed in the luciferase treated animals. Tumor growth repression T/C (Tumor/Control based on mean tumor volume) was plotted to day 19 (FIG. 8H) for each of the treatment groups. The four-component mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. exhibited the largest T/C.

[0901] Analogous to the study shown in FIG. 8, a study was conducted in which individual components were systematically removed from the mRNA mixture of IL-2, IL-12sc, GM-CSF and IFN.alpha. (ModB; SEQ ID NOs: 35, 41, 59, and 47). CT26 tumors received 6 intratumoral mRNA injections on days 19, 21, 23, 26, 28 and 30. Treatment with the cytokine mRNA mixture of IL-2, IL-12sc, GM-CSF and IFN.alpha. resulted in tumor regression in 4 of 10 animals led to complete tumor regression in 4 animals and stable disease beyond day 40 in two individuals (FIG. 9A). Tumors treated with the ModB 3-component mRNA mixtures of i) IL-2, IL-12sc, and IFN.alpha., ii) IL-2, GM-CSF and IFN.alpha., iii) IL-12sc, GM-CSF and IFN.alpha., and iv) IL-12sc, GM-CSF and IL-2 resulted in regression of 2, 3, 3 and 4 tumors, respectively (FIG. 9B-E) and CT26 tumors treated with a control luciferase mRNA displayed no tumor regression (FIG. 9F).

[0902] To analyze tumor growth kinetics, mean tumor volumes were calculated for each treatment group up to day 36. The smallest mean tumor volume was observed for mice treated with the mixture of IL-2, IL-12sc, GM-CSF and IFN.alpha., while the largest mean tumor volume was observed in the luciferase treated animals (FIG. 10 A). Tumor growth repression T/C (Tumor/Control based on mean tumor volume) was plotted to day 30 (FIG. 10B) for each of the treatment groups. The four-component mixture of IL-2, IL-12sc, GM-CSF and IFN.alpha. exhibited the largest T/C.

[0903] The anti-tumor response from the different cytokine mRNA mixtures in FIG. 9 were further analyzed as follows: The tumor volume change (.DELTA.V) as defined by the difference in tumor volume between the measurement at the end of experiment or termination (V.sub.t) and the measurement at the beginning of the experiment (V.sub.b) (.DELTA.V=V.sub.t-V.sub.b), was calculated for all mice in each of the six treatment groups, including mice treated with 4 component cytokine mRNA mixture (IL-2, IL-12sc, GM-CSF and IFN.alpha.), mice treated with 3 component mixture missing one of the four cytokines and mice treated with control luciferase mRNA. Mean (.mu.) and standard deviation (.sigma.) of a trimmed data (removing top and bottom 10% of the original data) on tumor volume change was calculated for control luciferase group and the Shapiro-Wilk normality test of this trimmed data showed it approximately followed a normal distribution. A Z score (Z=(.DELTA.V-.mu.)/.sigma.) was then calculated for tumor volume change data in each individual mouse from all treatment groups. A ratio (R) between each tumor volume change value and the trimmed mean of control group was calculated (R=.DELTA.V/.mu.). Given the definitions, we consider the Z score as "significance of tumor volume reduction" and R as "extent of tumor volume reduction". To identify a mouse that shows a significantly smaller tumor volume increase than that in control luciferase group (in other words "significant tumor reduction" as compared with control group), a cutoff of Z<=-1.645 (p(Z<=-1.645)=0.05) and R<=0.15 was applied. The results showed that the number of mice in each treatment group that has significantly smaller tumor volume increase than control group as follows: i) 6 out of 8 mice for the cytokine mRNA mixture of IL-2, IL-12sc, GM-CSF and IFN.alpha. (ModB). ii) 2 of 8 mice for IL-2, GM-CSF and IFN.alpha. (ModB), iii) 3 of 8 for IL-12sc, GM-CSF and IFN.alpha. and IL-2, IL-12sc, and IFN.alpha. and iv) 4 of 8 mice treated with the cytokine mRNA mixture of IL-2, IL-12sc, and GM-CSF (ModB) (FIG. 11).

[0904] Female C57BL/6J mice were implanted with B16F10 cells as described above. Mice were treated with 4 intratumoral injection (8 .mu.g mRNA/2 .mu.g per target) on days 11, 15, 19, and 23 with ModB cytokine mRNA mixture (IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha.) or control luciferase mRNA. Treatment with the 4-component mixture of cytokine mRNA resulted in tumor rejection in 6/10 treated mice. See, FIG. 24B. In comparison, no tumor free mice were observed in any of the groups treated with a single mRNA (FIGS. 24C-F). The combination of IL-15 sushi, IL-12sc, GM-CSF, IFN.alpha. led to increased overall survival with 60% of the mice tumor free at Day 70, while all tumors of mice treated with a single cytokine mRNA progressing to the stage where the animals needed to be euthanized (FIG. 24G). Luciferase control is shown in FIG. 24A.

Example 4--Cytokine mRNA Protects Against Tumor Re-Challenge

[0905] To evaluate the effect of the cytokine mRNA mixture on the development of immunological memory, re-challenge experiments were performed. Briefly, B16F10 tumor bearing mice were treated with a cytokine mRNA mixture of IL-15sushi, IL-12sc, GM-CSF, and IFN.alpha. (Mod B; SEQ ID NOs: 53, 41, 59, and 47). A portion of the cytokine mRNA treatment B16F10 tumors completely regressed leading to tumor free animals. These tumor free animals were then re-challenged with B16F10 cells as a way to assess adaptive immune memory and 9 naive mice were implanted with B16F10 tumor cells as a positive control for tumor engraftment (FIG. 12A). All 9 naive mice engrafted with B16F10 cells developed tumors, whereas all eight tumor-free mice rejected the B16F10 cells and did not exhibit growth of B16F10 tumors (FIG. 12B). A portion of mice previously treated with cytokine mRNA develop localized vitiligo at the original site of the tumor (FIG. 12C). This experiment was essentially repeated and results are shown in FIG. 33.

[0906] 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 IFN.alpha. (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-.DELTA.gp70) was knocked out. 9 and 10, respectively, naive mice were implanted with CT26 tumor cells and CT26-.DELTA.gp70 as a positive control for tumor engraftment. On day 21 after tumor inoculation 8 out of 9 naive mice had engrafted with CT26 tumor cells developed tumors and all 10 naive mice engrafted with CT26-.DELTA.gp70 tumor cells developed tumors whereas all three tumor-free mice in each group rejected the CT26 and CT26-.DELTA.gp70 cells and did not exhibit growth of CT26 tumors and CT26-.DELTA.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

[0907] 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 IFN.alpha. (ModB; SEQ ID NOs: 53, 41, 59, and 47). The right tumor was injected with mRNA at three different dose levels (80 .mu.g, 8 .mu.g, and 0.8 .mu.g mRNA corresponding to 20 .mu.g, 2 .mu.g and 0.2 .mu.g 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).

[0908] 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 IFN.alpha. (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. FIG. 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 FIG. 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; FIG. 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

[0909] 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 IFN.alpha.2b (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).

[0910] The pharmacodynamic effects of the human mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b 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 IFN.alpha.2b (ModB) or the individual cytokine mRNAs encoding IL-12sc, IFN.alpha.2b, 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 IFN.gamma. levels from 6 donors treated with the cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b was 5623 pg/mL, while treatment with the individual cytokine mRNA for IL-12sc, IFN.alpha.2b, IL-15 sushi or GM-CSF induced median IFN.gamma. levels of 534, 67, 17, and 4 pg/mL, respectively (FIG. 15B).

[0911] In vivo expression of the human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b (ModB) and IL-2, IL-12sc, GM-CSF and IFN.alpha.2b (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 IFN.alpha.2b (FIG. 16A), IL-2 (FIG. 16B), IL-12sc (FIG. 16C), IL-15 sushi (FIG. 16D), GM-CSF (FIG. 16E) were evaluated.

[0912] 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 IFN.alpha.2b (Table 4) and IL-2, IL-12sc, GM-CSF and IFN.alpha.2b (Table 5).

TABLE-US-00010 TABLE 4 Pharmacokinetic results for the IL-15 sushi mixture Tumor GM-CSF IFN.alpha.2b IL-12sc IL-15sushi t.sub.1/2 (hrs) 12.4 9.59 15.2 10.2 Tmax (hrs) 4 4 8 4 Cmax (pg/mg) 1591 4862 3767 2639

TABLE-US-00011 TABLE 5 Pharmacokinetic results for the IL-2 mixture Tumor GM-CSF IFN.alpha.2b IL-12sc IL-2 t.sub.1/2 (hrs) 14.8 10.2 15.5 12.8 Tmax (hrs) 2 2 8 2 Cmax (pg/mg) 2487 10685 4961 4271

[0913] Induction of the human interferon alpha genes ISG15, ISG54 and MX1 were monitored in the A375 tumors as a pharmacodynamics marker at 2 h, 4 h, 8 h, 24 h, 48 h and 72 h following mRNA injection of the cytokine mRNA mixtures of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha.2b (ModB) and IL-2, IL-12sc, GM-CSF and IFN.alpha.2b (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

[0914] 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 IFN.alpha. (SEQ ID NO: 44) or ModB mRNA encoding IFN.alpha. (SEQ ID No: 47) was added to the ModA or ModB mRNA mixes, respectively (FIGS. 18D and 18E). Anti-tumor activity was assessed.

[0915] Mice treated with this combination of ModA mRNA had 4/9 mice tumor-free without IFN.alpha. (FIG. 18B) and 3/9 mice tumor-free with IFN.alpha. (FIG. 18D). Therefore, treatment with IFN.alpha. mRNA did not appear to increase the response to the cytokines when mRNA was dosed in the ModA form.

[0916] In contrast, mice treated with the combination of ModB mRNA had 1/9 mice tumor-free without IFN.alpha. (FIG. 18C) and 7/9 mice tumor-free with IFN.alpha. (FIG. 18E). Thus, treatment with IFN.alpha. 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

[0917] 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 IFN.alpha. (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 5 mg/kg) on Days 10, 13, 16, 19, 22, 25. Groups were as follows: 1) control mRNA (80 .mu.g total mRNA; 50 .mu.L intratumoral injection at 1.6 mg/mL plus control isotype antibody (clone MOPC-21 (BioLegend); 5 mg/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.

[0918] 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 IFN.alpha. (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 .mu.g total mRNA; 50 .mu.L 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.

[0919] 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 IFN.alpha. (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 .mu.g/200 .mu.L per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 .mu.g/200 .mu.L 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.

[0920] 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 IFN.alpha. (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 .mu.g/200 .mu.L per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 .mu.g/200 .mu.L 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

[0921] 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 .mu.g (2 .mu.g/target) human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN.alpha. (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 IFN.alpha. (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

[0922] 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 IFN.alpha. (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 .mu.g (20 .mu.g) to the lowest dose of 0.0256 .mu.g (0.0064 .mu.g). Results are shown in FIGS. 23A-D.

Example 11--Cytokine mRNA Leads to Expansion of Gp70+CD8 T Cells

[0923] 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, IFN.alpha., 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

[0924] Mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of 80 .mu.g of mRNA (20 .mu.g/target) into right tumor which was initiated with 0.5.times.10{circumflex over ( )}6 cells (treated), while the tumor initiated with 0.25.times.10{circumflex over ( )}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, IFN.alpha., 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.

[0925] 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).

[0926] 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.

[0927] Relative abundance of infiltrated immune cells is determined by calculating the average expression of immune cell-type specific gene signatures.

TABLE-US-00012 TABLE 6 Treated_cytokine Treated_cytokine Treated_cytokine Untreated_cytokine Untreated_cytokine Untreated_cytokine mRNA mix/IgG1 mRNA mix/IgG1 mRNA mix/IgG1 mRNA mix/IgG1 mRNA mix/IgG1 mRNA mix/IgG1 IFNG_reg- vs. Luc mRNA/ vs Luc mRNA/ vs Luc mRNA/ vs Luc mRNA/ vs Luc mRNA/ vs Luc mRNA/ Gene ulation IgG1.foldChange IgG1.RawPValue IgG1.FDR_BH IgG1.FoldChange IgG1.RawPValue IgG1.FDR_BH ABI1 Activate 1.6776 0.0073 0.0995 1.5218 0.0354 0.1551 ACOD1 Activate 31.6293 3.9652E-06 0.0024 6.1017 0.0096 0.0911 ADGRG2 Activate 1.8174 0.0248 0.2076 1.3902 0.2293 0.4662 ADORA2B Activate 2.9657 0.0044 0.0752 1.4261 0.352 0.6138 AIF1 Activate 6.0406 0.0005 0.0201 4.7598 0.0032 0.057 AIM2 Activate 9.9707 0.000085324 0.0086 3.4835 0.0276 0.1361 ANGPTL4 Activate 2.0707 0.0293 0.2265 2.0904 0.0368 0.1586 APOL6 Activate 10.3994 0.0017 0.0418 10.1341 0.0031 0.0564 APP Activate 2.0312 0.0006 0.0238 2.2382 0.0003 0.0276 ARFGAP3 Activate 1.6535 0.0146 0.1517 1.0201 0.9237 1 ASS1 Activate 3.5414 0.0084 0.1093 -1.0202 0.9668 1 ATF3 Activate 5.5284 0.0006 0.0231 -1.1007 0.8409 1 B2M Activate 4.7005 0.000092097 0.0089 2.7946 0.0085 0.0859 BACH1 Activate 2.2944 0.0098 0.1179 1.3281 0.3821 0.6482 BATF2 Activate 4.0479 0.0211 0.1885 1.6468 0.4201 0.6889 BCL2L11 Activate 4.2973 0.0057 0.0868 1.1465 0.7946 1 BCL3 Activate 4.4411 0.0167 0.1639 2.5676 0.1413 0.3455 BLNK Activate 4.2753 0.0003 0.0158 3.2008 0.0044 0.0643 BST1 Activate 16.9081 1.3371E-06 0.0016 6.6164 0.0007 0.0334 BTG1 Activate 3.5307 0.0001 0.0102 -1.1775 0.5968 0.8607 C1QA Activate 5.3999 0.0029 0.0588 1.3506 0.5911 0.8558 C1QB Activate 5.955 0.0104 0.1219 3.531 0.0786 0.2408 C1QC Activate 6.2282 0.0009 0.0307 2.7013 0.0707 0.2284 C2 Activate 2.2866 0.0367 0.2598 2.5908 0.0242 0.1281 C3 Activate 10.2057 0.0043 0.0734 11.6428 0.0044 0.0648 C4B Activate 3.3143 0.0154 0.1562 1.3973 0.5045 0.7768 C5AR1 Activate 6.4343 0.000061281 0.0074 1.8122 0.1729 0.3925 CAMK4 Activate 2.5261 0.0011 0.0335 2.0726 0.012 0.1009 CASP1 Activate 8.2891 0.0079 0.1049 6.7759 0.0214 0.1263 CASP4 Activate 3.9281 0.0015 0.0389 1.2492 0.5969 0.8608 CCL11 Activate 6.225 0.0028 0.0579 9.5207 0.0007 0.0334 CCL17 Activate 3.9738 0.0268 0.2146 3.0432 0.0868 0.2549 CCL2 Activate 5.0444 0.0331 0.2446 1.9159 0.4045 0.6716 CCL22 Activate 5.4223 0.000050981 0.0069 3.6151 0.002 0.0467 CCL3 Activate 8.2215 0.0007 0.0259 4.7966 0.0129 0.1045 CCL4 Activate 8.7234 0.0008 0.0287 2.8285 0.1042 0.2846 CCL5 Activate 19.1375 0.000004935 0.0027 9.4224 0.0004 0.0299 CCL7 Activate 4.7053 0.0068 0.0952 -1.2497 0.6964 0.9457 CCL8 Activate 9.3589 0.0113 0.1283 3.9524 0.1286 0.3243 CCR1 Activate 5.333 0.0012 0.0349 1.7221 0.2823 0.5325 CCR2 Activate 5.8007 0.0025 0.0546 2.1931 0.1766 0.3975 CCR5 Activate 10.8409 0.000010504 0.004 4.8697 0.0025 0.0512 CCRL2 Activate 3.9822 0.002 0.0465 3.6428 0.0056 0.071 CD14 Activate 5.6413 0.0025 0.0546 1.413 0.5406 0.8104 CD2 Activate 13.539 0.0001 0.0095 5.0981 0.0138 0.1086 CD274 Activate 11.0684 0.000096803 0.0091 3.4411 0.038 0.1616 CD38 Activate 5.5697 0.0005 0.0212 1.4645 0.4221 0.6908 CD40 Activate 11.0285 0.0003 0.0158 1.2537 0.7201 0.9637 CD40LG Activate 3.1168 0.0068 0.0952 1.1976 0.6673 0.9224 CD68 Activate 6.1325 0.000040468 0.0063 3.6801 0.0029 0.0544 CD74 Activate 30.4664 0.000057089 0.0072 5.9291 0.0291 0.1392 CD80 Activate 4.1003 0.0005 0.0217 3.5579 0.0026 0.0517 CD86 Activate 6.3927 0.0008 0.027 8.2257 0.0004 0.0289 CDKN2A Activate 1.9017 0.0117 0.1312 -1.2679 0.3579 0.6202 CEBPB Activate 4.4389 0.0007 0.0253 2.1429 0.0784 0.2404 CELSR1 Activate 1.6953 0.0001 0.0107 1.1455 0.3001 0.5534 CERS6 Activate 2.2538 0.0064 0.0927 1.717 0.0763 0.2363 CFB Activate 9.003 0.0001 0.0109 6.2886 0.0018 0.0444 CH25H Activate 4.3757 0.007 0.097 1.9042 0.2435 0.4858 CHRNE Activate 1.6587 0.0293 0.2263 1.0536 0.8254 1 CIITA Activate 6.2751 0.0008 0.0287 2.736 0.0651 0.2172 CLEC2D Activate 4.9639 0.0002 0.0135 2.6567 0.0223 0.1263 CLEC4E Activate 25.396 2.9823E-07 0.001 7.0201 0.0009 0.0355 CLIC4 Activate 2.2909 0.000025847 0.0053 1.4654 0.0399 0.1658 CMPK2 Activate 2.6988 0.0048 0.0785 1.9213 0.0687 0.2249 CORO1A Activate 5.8653 0.0076 0.1019 2.1612 0.2494 0.4929 CP Activate 2.7012 0.0222 0.1945 1.2703 0.5885 0.8544 CSF1 Activate 5.3648 0.0071 0.0977 2.9314 0.0916 0.2624 CSF2 Activate 2.4249 0.0053 0.0828 1.8261 0.0635 0.2145 CSF2RB Activate 7.8798 0.000014063 0.0041 3.3417 0.008 0.0833 CTSB Activate 2.9396 0.0192 0.1786 -1.5854 0.3271 0.5867 CTSD Activate 1.7662 0.0058 0.0873 1.5654 0.0351 0.1546 CTSH Activate 6.6723 0.0004 0.0189 3.0998 0.0341 0.1523 CTSS Activate 14.2913 0.000052305 0.0069 2.0554 0.2402 0.4813 CXCL1 Activate 2.4631 0.0443 0.2909 2.3701 0.0679 0.223 CXCL10 Activate 4.0615 0.0183 0.174 3.84 0.0314 0.1453 CXCL11 Activate 6.3825 0.0032 0.0623 2.1745 0.2167 0.4494 CXCL12 Activate 4.1992 0.0099 0.1191 3.9497 0.0188 0.1263 CXCL16 Activate 11.7476 0.000012898 0.0041 2.7724 0.052 0.1919 CXCL2 Activate 17.8737 0.000065478 0.0076 2.0435 0.2897 0.5414 CXCL3 Activate 2.5623 0.0157 0.1584 -1.1159 0.7808 1 CXCL9 Activate 10.5652 0.002 0.0475 6.7433 0.0152 0.1139 CYBB Activate 10.2429 0.0001 0.0102 2.4115 0.1288 0.3246 CYLD Activate 2.1952 0.0005 0.0217 1.0366 0.8685 1 CYSLTR2 Activate 4.6876 0.0015 0.0389 -1.3886 0.4896 0.7624 DAPK1 Activate 2.9566 0.0202 0.1837 2.3734 0.0752 0.2341 DAXX Activate 2.5923 0.0255 0.2101 2.7152 0.0271 0.1349 DDIT3 Activate 2.2027 0.0286 0.2233 2.4229 0.0212 0.1263 DDX58 Activate 2.2084 0.0054 0.084 1.2566 0.422 0.6908 DPP4 Activate 4.4101 0.047 0.3002 2.2805 0.2869 0.5383 DTX3L Activate 3.4716 0.000027857 0.0053 1.6387 0.0759 0.2356 EBI3 Activate 5.2118 0.0003 0.0153 2.8451 0.0203 0.1263 ECE1 Activate 3.9089 0.0033 0.0624 1.8673 0.1782 0.3996 EGLN3 Activate 2.9572 0.0465 0.2989 1.0391 0.9453 1 EGR2 Activate 2.4479 0.0155 0.157 2.5764 0.0158 0.1159 ERAP1 Activate 2.5669 0.0033 0.0634 2.2811 0.0134 0.1068 FAM26F Activate 14.7052 0.0008 0.0279 26.6887 0.0002 0.0227 FAS Activate 7.0896 0.0003 0.0169 4.7216 0.0052 0.0695 FCER1G Activate 6.7045 0.0007 0.0254 4.8631 0.0059 0.0728 FGF2 Activate 2.9272 0.0257 0.2106 1.8257 0.2249 0.4606 FGL2 Activate 12.456 9.3027E-06 0.0038 4.9116 0.0036 0.0599 FPR2 Activate 26.1833 0.000096227 0.0091 4.1758 0.0744 0.2328 FTH1 Activate 1.6855 0.0413 0.2791 2.2166 0.0045 0.0657 GBP2 Activate 7.2783 0.0007 0.0248 6.7229 0.0017 0.0437 GBP3 Activate 6.2795 0.0006 0.0222 3.0356 0.0358 0.156 GBP4 Activate 18.7632 0.0002 0.0139 6.7981 0.0154 0.1146 GBP5 Activate 18.254 0.0003 0.0157 7.5534 0.0118 0.1 GBP6 Activate 6.4537 0.0004 0.0187 8.0969 0.0002 0.0266 GBP7 Activate 4.3285 0.0017 0.0427 2.3295 0.0713 0.2296 GCH1 Activate 3.9933 0.0057 0.0867 -2.2318 0.1144 0.3009 GNA13 Activate 1.843 0.0129 0.1403 1.2404 0.3874 0.6539 GSDMD Activate 5.5069 0.000067203 0.0076 4.0075 0.0013 0.0403 GZMB Activate 10.9342 0.001 0.0323 11.0118 0.0018 0.0442 H2-M3 Activate 6.3613 0.0003 0.0169 -1.2831 0.6129 0.8754 H2-Q7 Activate 13.035 0.00007123 0.0078 13.2107 0.0001 0.0209 H2-T23 Activate 9.9835 0.0035 0.0651 1.093 0.9091 1 HCAR2 Activate 5.1405 0.0007 0.0254 3.0731 0.0206 0.1263 HCK Activate 7.0952 0.000025322 0.0053 4.1767 0.0019 0.0455 HIF1A Activate 1.661 0.0261 0.2122 1.117 0.6345 0.8942 HMOX1 Activate 2.9191 0.0049 0.0797 2.2892 0.0345 0.1532 ICAM1 Activate 5.8185 0.0005 0.0217 4.9939 0.0023 0.0487 ICOSL Activate 1.9545 0.0086 0.1107 1.8592 0.0203 0.1263 IDO1 Activate 2.1676 0.007 0.097 1.5151 0.1535 0.3644 IFI27 Activate 2.1198 0.0058 0.088 1.1685 0.5669 0.8345 IFI44 Activate 10.2739 0.0001 0.0093 5.8893 0.0032 0.057 IFIH1 Activate 3.3922 0.0001 0.0093 1.8886 0.0369 0.1588 IFIT1 Activate 4.7651 0.000038724 0.0062 2.3488 0.0191 0.1263 IFIT2 Activate 2.6547 0.0381 0.266 1.6098 0.3269 0.5865 IFIT3 Activate 6.6433 0.0003 0.0153 2.3076 0.0981 0.2746 IFITM1 Activate 13.8818 0.0017 0.0427 4.0409 0.096 0.2703 IFITM3 Activate 5.3416 0.000049885 0.0069 4.6318 0.0003 0.0278 IFNG Activate 15.8295 0.000030559 0.0055 5.3984 0.0086 0.0862 IFNGR1 Activate 4.6529 0.0003 0.0169 1.1624 0.713 0.9579 IKBKE Activate 3.6191 0.000070796 0.0078 2.1509 0.0151 0.1135 IL10RA Activate 6.6362 0.0006 0.0225 2.2744 0.1259 0.3198 IL12B Activate 1.5869 0.0237 0.2019 1.3356 0.1698 0.3882 IL12RB1 Activate 3.6563 0.0032 0.0623 2.7374 0.0261 0.1323 IL12RB2 Activate 10.8737 0.0001 0.0105 2.7693 0.0898 0.2596 IL15RA Activate 5.6222 0.0002 0.0124 3.263 0.01 0.0929 IL18BP Activate 13.3799 0.000016505 0.0045 2.6518 0.0808 0.2445 IL18R1 Activate 10.5794 0.0004 0.0172 4.1773 0.0288 0.1387 IL18RAP Activate 12.8979 1.6368E-06 0.0016 3.9032 0.0058 0.0721 IL1A Activate 5.0611 0.0002 0.014 2.016 0.1006 0.2792 IL1B Activate 7.1785 0.0017 0.0427 2.9628 0.0846 0.2505 IL1RL1 Activate 3.2501 0.0421 0.2825 2.7191 0.0999 0.2778 IL1RN Activate 11.4696 0.000016637 0.0045 3.5493 0.0183 0.1256 IL27 Activate 5.0377 0.0003 0.0145 2.9655 0.0136 0.1077 IL2RA Activate 12.629 0.000036735 0.0059 9.0232 0.0004 0.03 IL3RA Activate 2.0071 0.0463 0.2977 -2.3554 0.0222 0.1263 INHBA Activate 7.0079 0.0006 0.0238 8.1176 0.0005 0.0316 IRF1 Activate 4.8896 0.0002 0.0136 4.2429 0.0011 0.0383 IRF2 Activate 3.0856 0.0001 0.0102 2.3475 0.0038 0.0609 IRF4 Activate 3.6001 0.0009 0.0303 2.7396 0.0107 0.0955 IRF5 Activate 6.3959 0.0039 0.0697 6.2724 0.0066 0.0767 IRF7 Activate 4.6951 0.0016 0.0408 2.6708 0.0468 0.1806 IRGM1 Activate 3.5604 0.0205 0.1855 3.8887 0.0196 0.1263 ISG15 Activate 6.1635 0.0003 0.0158 2.64 0.0488 0.1845 ISG20 Activate 3.3567 0.0012 0.0349 3.9719 0.0006 0.0322 ITGA4 Activate 2.0304 0.0397 0.272 2.0083 0.055 0.1984 ITGAL Activate 16.3545 0.000032377 0.0056 6.1759 0.0055 0.0707 ITGAM Activate 8.5704 0.0093 0.1151 3.5033 0.1371 0.3385 ITGAX Activate 3.9189 0.0101 0.1202 2.0482 0.185 0.4084 ITGB2 Activate 7.7479 0.0002 0.0124 6.9547 0.0006 0.0332 JAK2 Activate 1.6647 0.0461 0.2976 1.3678 0.2378 0.4779 JUN Activate 1.7589 0.0035 0.0649 -1.1739 0.4037 0.6708 KLF4 Activate 1.907 0.0409 0.2778 1.156 0.6546 0.9116 KLF6 Activate 2.4625 0.0037 0.0672 2.1351 0.0182 0.125 KYNU Activate 3.8871 0.008 0.1057 4.8859 0.0039 0.061 LAG3 Activate 7.4221 0.0002 0.0114 2.156 0.1298 0.3262 LAT2 Activate 2.6441 0.028 0.2208 1.5322 0.3475 0.6094 LCN2 Activate 4.3524 0.0296 0.2275 16.3586 0.0003 0.0276 LCP2 Activate 8.4235 0.0004 0.0193 4.07 0.0205 0.1263 LGALS9 Activate 1.6825 0.0051 0.0817 1.6728 0.0085 0.086 LST1 Activate 7.6841 0.02 0.1832 1.6707 0.5648 0.8328 LTA Activate 2.7386 0.0055 0.0851 2.4968 0.0157 0.1157 LTB Activate 8.1723 0.0014 0.0379 2.3655 0.1846 0.4079 LY6E Activate 5.1119 0.0028 0.0572 2.8148 0.061 0.2104 LY96 Activate 2.4218 0.0302 0.2305 1.5311 0.3103 0.5661 LYN Activate 5.1219 0.001 0.0325 1.6841 0.2851 0.536 MAP3K8 Activate 7.131 0.0009 0.0302 1.0205 0.9717 1 MEFV Activate 10.8378 0.000019758 0.0047 2.3756 0.0951 0.2688 MMP12 Activate 6.4662 0.0035 0.0647 5.0068 0.015 0.1131 MX1 Activate 6.1489 0.0128 0.1395 2.6469 0.1916 0.4171 MX2 Activate 3.9804 0.0005 0.0205 1.8666 0.1068 0.2889 NAMPT Activate 2.341 0.0066 0.0943 -1.4194 0.2673 0.514 NAPSA Activate 4.3452 0.0003 0.0149 4.7434 0.0003 0.0272 NEURL3 Activate 5.943 0.0009 0.0291 1.9354 0.2066 0.4363 NFKB1 Activate 1.8089 0.0051 0.0815 1.0738 0.7351 0.9751 NFKBIA Activate 4.9409 0.000013016 0.0041 2.4668 0.0097 0.0912 NFKBIZ Activate 2.328 0.0073 0.0995 2.0257 0.0308 0.1436 NGF Activate 3.1687 0.0065 0.0933 3.4968 0.0055 0.0706 NLRC5 Activate 3.4173 0.0109 0.1261 2.2706 0.0985 0.2752 NLRP3 Activate 5.11 0.0007 0.0254 3.4525 0.0111 0.0975 NMI Activate 2.0435 0.0228 0.1979 1.6117 0.1416 0.3459 NOD2 Activate 3.5157 0.000018135 0.0045 2.5449 0.0013 0.0396 NOS2 Activate 26.5216 2.2445E-06 0.0017 24.8128 7.9556E-06 0.0117 NOTCH1 Activate 2.2952 0.0301 0.2303 1.4949 0.3078 0.5625 NR1D1 Activate 1.7995 0.0049 0.0799 1.8784 0.0045 0.065 NUPR1 Activate 2.8421 0.001 0.0318 1.7353 0.0807 0.2444 OAS2 Activate 3.8997 0.0003 0.0154 2.3682 0.0204 0.1263 OAS3 Activate 14.5317 7.7153E-06 0.0034 4.0924 0.0123 0.1019 P2RY14 Activate 16.6314 0.0005 0.0204 4.8513 0.0471 0.1812 P2RY6 Activate 5.352 0.0001 0.0103 2.2094 0.0606 0.2093 PARP9 Activate 3.1267 0.0179 0.1716 -1.1573 0.7644 0.9977 PARVG Activate 6.3047 0.0025 0.0539 1.2154 0.7444 0.9822 PDCD1LG2 Activate 11.6201 2.1928E-06 0.0017 3.9612 0.0045 0.065 PF4 Activate 6.7372 0.0284 0.2226 -2.2437 0.3655 0.6285 PIM1 Activate 4.0708 0.0003 0.0169 2.0732 0.0575 0.2034 PLA2G16 Activate 4.8619 0.001 0.0309 1.3802 0.4886 0.7613 PLAU Activate 3.8214 0.00003401 0.0058 2.1662 0.0131 0.1056 PLEK Activate 5.9274 0.0049 0.0805 2.9561 0.0921 0.2632 PMAIP1 Activate 3.4224 0.0002 0.0134 2.6199 0.0041 0.0627 PRDM1 Activate 3.5702 0.0297 0.2283 4.0335 0.0251 0.13 PRKCD Activate 4.5574 2.2524E-07 0.001 2.0325 0.0066 0.0768 PRKCQ Activate 2.1379 0.0065 0.0932 2.1615 0.0089 0.087 PSMB10 Activate 3.6197 0.005 0.0807 1.4724 0.3976 0.6643 PSMB8 Activate 3.9093 0.0032 0.0615 2.652 0.0388 0.1637 PSMB9 Activate 7.4132 0.0005 0.0197 4.5308 0.009 0.0877 PSME1 Activate 6.1609 0.0001 0.0096 3.9584 0.0037 0.06 PTAFR Activate 8.5218 0.000052564 0.0069 2.2614 0.1024 0.2821 PTGES Activate 3.3225 0.0241 0.2039 1.61 0.3819 0.6479 PTGS2 Activate 10.9036 0.0075 0.1017 3.9538 0.1309 0.3282 PTX3 Activate 5.271 0.0253 0.2091 1.8833 0.4052 0.672 RAB20 Activate 2.29 0.0257 0.2106 1.1261 0.7537 0.9893 RAC2 Activate 7.9519 0.000098427 0.0091 3.7008 0.0123 0.1021 RIPK1 Activate 1.6822 0.0456 0.2959 -1.1344 0.6373 0.8964 RSAD2 Activate 3.916 0.0003 0.0152 1.6141 0.184 0.407 RTP4 Activate 4.8033 0.0025 0.0537 2.5839 0.0696 0.2269 RUNX2 Activate 5.3191 0.0001 0.0095 2.2697 0.0491 0.185 RUNX3 Activate 5.5652 0.0012 0.0354 5.5875 0.002 0.0467 S100A10 Activate 2.1419 0.001 0.0318 -1.0647 0.7799 1 S100A8 Activate 10.9785 0.0065 0.0931 11.8118 0.0079 0.0832 S100A9 Activate 5.1245 0.0058 0.0879 4.0622 0.0228 0.1263 S1PR3 Activate 2.8084 0.0426 0.2844 4.3054 0.0083 0.0851 SAMHD1 Activate 6.8165 0.000028539 0.0053 5.2676 0.0004 0.0289 SELL Activate 9.0266 0.000036704 0.0059 2.6848 0.0503 0.1879 SEMA4A Activate 4.5461 0.0002 0.0134 7.1376 0.000014188 0.0118 SEPT3 Activate 1.9813 0.0021 0.0484 1.2827 0.2581 0.5036

SERPINB9 Activate 3.6968 0.0068 0.0955 1.3544 0.5306 0.8014 SERPINE1 Activate 2.7348 0.0131 0.142 1.0382 0.9268 1 SERPING1 Activate 8.5956 0.0003 0.0154 5.6206 0.0042 0.063 SHARPIN Activate 1.8222 0.0003 0.0169 -1.1856 0.2897 0.5414 SLAMF1 Activate 4.2282 0.000066073 0.0076 1.8422 0.0753 0.2344 SLC11A1 Activate 8.1631 0.0002 0.0114 6.6594 0.0009 0.0364 SLC15A3 Activate 13.0122 0.0001 0.0093 4.2688 0.0238 0.1278 SLC16A9 Activate 2.2466 0.0273 0.2172 1.6072 0.21 0.4406 SLC28A2 Activate 6.903 0.0001 0.0096 1.4304 0.4466 0.7176 SLFN5 Activate 3.7058 0.0022 0.0493 2.1361 0.0772 0.2383 SMAD7 Activate 3.1247 0.0209 0.187 -1.1526 0.7767 1 SOCS1 Activate 3.9437 0.011 0.1267 3.3365 0.0325 0.1481 SOD2 Activate 2.6759 0.0047 0.0774 3.1489 0.0021 0.0475 SOD3 Activate 2.1257 0.0158 0.1591 1.4255 0.2663 0.5128 SP100 Activate 4.8282 7.0055E-06 0.0032 2.8699 0.0019 0.0452 SP110 Activate 3.7976 0.0057 0.0867 1.1957 0.71 0.9556 SPI1 Activate 10.5793 0.000055966 0.0071 6.5856 0.0014 0.0409 SPP1 Activate 2.5268 0.0212 0.1893 2.4021 0.0384 0.1624 STAT1 Activate 4.2736 0.0089 0.1122 3.9844 0.0176 0.1229 STAT2 Activate 5.4847 0.000013182 0.0041 3.019 0.0035 0.0589 STAT4 Activate 6.3819 0.0038 0.069 1.1576 0.8175 1 STAT6 Activate 1.7399 0.0097 0.1171 1.7712 0.0116 0.0993 STX11 Activate 3.6303 0.0058 0.088 1.7593 0.2306 0.4679 TAP1 Activate 4.588 0.001 0.0323 2.7092 0.0329 0.1493 TAP2 Activate 2.8268 0.0148 0.1526 1.5315 0.3257 0.585 TAPBP Activate 2.5416 0.0274 0.2176 1.7971 0.1801 0.4019 TAPBPL Activate 3.0796 0.0031 0.0609 2.4014 0.0251 0.13 TBX21 Activate 3.8788 0.0008 0.0273 4.1566 0.0008 0.0354 TCIRG1 Activate 2.7899 0.0005 0.0217 1.2508 0.4321 0.7008 TGFBR2 Activate 1.9044 0.031 0.2346 1.2596 0.451 0.7222 THBS1 Activate 2.8135 0.005 0.0807 2.9522 0.0055 0.0707 THEMIS2 Activate 9.3368 0.0011 0.0339 6.5099 0.0079 0.0832 THY1 Activate 5.5745 0.0036 0.0666 3.0999 0.0597 0.2073 TLR1 Activate 3.9534 0.0044 0.0751 4.9709 0.002 0.0463 TLR2 Activate 4.2854 0.0004 0.0173 1.0613 0.8785 1 TLR7 Activate 3.6076 0.0317 0.2384 1.1789 0.7874 1 TLR8 Activate 7.9426 0.0012 0.0349 1.5453 0.4846 0.7574 TLR9 Activate 7.0125 0.0004 0.0182 5.5003 0.0026 0.052 TMEM50B Activate 2.2646 0.0029 0.0584 1.6005 0.089 0.2584 TNF Activate 15.5761 0.000001549 0.0016 3.6025 0.014 0.1094 TNFAIP2 Activate 4.2629 0.0043 0.0737 1.3184 0.5837 0.8504 TNFRSF11A Activate 3.7342 0.0089 0.1124 3.2927 0.0236 0.1273 TNFRSF14 Activate 3.3507 0.0151 0.1547 2.0443 0.1615 0.3761 TNFRSF1B Activate 6.9767 0.0004 0.0187 3.0014 0.0433 0.1723 TNFSF10 Activate 5.9466 0.0008 0.0286 2.8254 0.0505 0.1883 TNFSF12 Activate 4.0396 0.0003 0.0145 3.0636 0.0039 0.061 TNFSF13 Activate 3.7405 0.0229 0.1979 -1.7547 0.3434 0.6048 TNFSF13B Activate 4.2939 0.00009167 0.0089 1.0175 0.96 1 TRAFD1 Activate 7.5123 0.000066493 0.0076 1.4364 0.4415 0.7113 TRIM21 Activate 3.1067 0.0069 0.096 -1.1129 0.7984 1 TXK Activate 3.1599 0.0017 0.0418 1.8261 0.0992 0.2766 UBD Activate 39.4489 3.1101E-06 0.0021 20.5238 0.0001 0.0203 UBE2L6 Activate 3.418 0.002 0.0464 4.4962 0.0005 0.031 USP18 Activate 5.4886 0.0004 0.0192 1.697 0.258 0.5036 VCAM1 Activate 7.3103 0.0003 0.0163 1.2409 0.6812 0.9337 WARS Activate 1.5033 0.0098 0.1179 1.8802 0.0003 0.0284 ZFP36 Activate 3.0067 0.0051 0.0815 -1.1887 0.6585 0.9153 AHCY Inhibit -2.0639 0.0374 0.2628 -1.8643 0.0878 0.2566 AQP1 Inhibit -2.6893 0.0012 0.0354 -2.9843 0.0008 0.0346 AZGP1 Inhibit -3.6306 0.0039 0.0701 -3.72 0.0054 0.0705 CSE1L Inhibit -1.9528 0.0448 0.2931 -4.9001 0.000053442 0.0177 DHX9 Inhibit -1.9049 0.0192 0.1787 -1.3891 0.2439 0.4863 EMID1 Inhibit -2.5566 0.047 0.3003 -1.9661 0.1701 0.3887 FKBP6 Inhibit -4.1867 0.0043 0.0733 -2.2618 0.1075 0.2898 FLT4 Inhibit -1.7478 0.0248 0.2076 -1.6367 0.0588 0.2058 GMPR Inhibit -2.9853 0.027 0.2154 -5.5877 0.0017 0.0432 GNAO1 Inhibit -2.2497 0.0179 0.1721 -1.0647 0.8565 1 HSP90AB1 Inhibit -1.9951 0.007 0.0974 -1.1404 0.6083 0.8715 IDI1 Inhibit -2.9218 0.004 0.0713 -2.402 0.023 0.1263 IGFBP4 Inhibit -3.3872 0.0211 0.1885 -3.0044 0.0473 0.1815 MIF Inhibit -3.5707 0.0052 0.0828 -1.3189 0.5425 0.8124 MSH2 Inhibit -1.909 0.0328 0.2435 -1.7932 0.0664 0.2198 MYC Inhibit -1.8764 0.0225 0.1962 -1.5059 0.1507 0.36 PBK Inhibit -4.8198 0.0008 0.0287 -10.4275 0.000012076 0.0117 PCDH17 Inhibit -2.2868 0.0029 0.0588 -1.1447 0.6223 0.8835 PLCG1 Inhibit -1.6389 0.0439 0.2895 1.263 0.3564 0.6189 POLR2F Inhibit -1.7137 0.0139 0.1473 -2.1058 0.0019 0.0456 POMP Inhibit -1.5182 0.047 0.3002 -1.3884 0.1354 0.336 PRPF8 Inhibit -2.0788 0.0159 0.1593 -2.1397 0.0179 0.1239 PTGES2 Inhibit -1.9137 0.0278 0.2201 -1.0746 0.811 1 RAD18 Inhibit -2.1129 0.0032 0.0615 -1.7251 0.0354 0.1551 SF3A1 Inhibit -1.598 0.0042 0.0729 -1.2281 0.2111 0.4418 SLC12A2 Inhibit -2.8366 0.0039 0.0697 -3.5987 0.0011 0.0373 SMTN Inhibit -1.8432 0.0398 0.2725 -1.2994 0.3927 0.6593 SQLE Inhibit -4.0941 0.0396 0.2717 -4.8892 0.0296 0.1404 SREBF2 Inhibit -2.3929 0.0032 0.0615 -3.5164 0.0001 0.0209 SSBP1 Inhibit -1.5752 0.0094 0.1156 1.0074 0.9665 1 TMEM158 Inhibit -1.6002 0.044 0.2897 1.4394 0.1352 0.3357 TYMP Inhibit -2.6432 0.0448 0.2928 1.469 0.4412 0.7111 UNC5B Inhibit -2.3093 0.0267 0.2143 -1.5396 0.2666 0.5132

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

[0928] Mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of 80 .mu.g of mRNA (20 .mu.g/target) into right tumor which was initiated with 0.5.times.10{circumflex over ( )}6 cells only one of the tumors (treated), while the other tumor initiated with 0.25.times.10{circumflex over ( )}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 FIGS. 28A and 28B were treated with cytokine mRNA, while mice from tumors in FIGS. 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 FIGS. 28E, F, G. The results indicate that a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN.alpha., 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

[0929] Mice bearing a single B16F10 tumor received a single intratumoral injection with mRNA encoding the Thy1.1 protein (FIG. 29A-G). Approximately 18 hrs 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

[0930] Mice bearing B16F10 tumors received a single mRNA injection with 80, 8 or 0.8 .mu.g of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN.alpha., 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, IFN.alpha., and IL-12sc, IFNgamma and IP-10 were quantified in the tumor lysate. FIGS. 30A-F show that the cytokine mRNA was expressed intratumorally in a dose-dependent manner.

[0931] 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, IFN.alpha., 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).

[0932] 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, IFN.alpha., 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

[0933] Mice bearing B16F10 tumors received a single intratumoral injection of 50 .mu.g 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. FIGS. 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

[0934] Groups of mice bearing B16F10 tumors were treated with 100 .mu.g 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 .mu.g cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN.alpha., 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 FIG. 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

[0935] WT C57BL6J mice and C57BL6J mice deficient for the murine IFN.gamma. (IFN.gamma. KO) were implanted with B16F10 tumor cells as described in Example 1. Mice were treated by intratumoral injection with 80 .mu.g (20 .mu.g/target) cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN.alpha., and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47) or 80 .mu.g control mRNA, and overall survival was monitored. The results, depicted in FIG. 35, indicate that mice lacking IFN.gamma. did not exhibit a detectable antitumor response when treated with the cytokine mRNA.

Sequence CWU 1

1

88152DNAArtificial sequenceModA 5' UTR 1gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca cc 52252RNAArtificial sequenceModA 5' UTR 2gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca cc 523145DNAArtificial sequenceModB 5' UTR 3ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagcc 1454145RNAArtificial sequenceModB 5' UTR 4ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagcc 145552DNAArtificial sequenceAlternative Mod 5' UTR 5agacgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca cc 52652RNAArtificial sequenceAlternative Mod 5' UTR 6agacgaacua guauucuucu gguccccaca gacucagaga gaacccgcca cc 527427DNAArtificial sequenceModA/B 3' UTR 7ctcgagctgg tactgcatgc acgcaatgct agctgcccct ttcccgtcct gggtaccccg 60agtctccccc gacctcgggt cccaggtatg ctcccacctc cacctgcccc actcaccacc 120tctgctagtt ccagacacct cccaagcacg cagcaatgca gctcaaaacg cttagcctag 180ccacaccccc acgggaaaca gcagtgatta acctttagca ataaacgaaa gtttaactaa 240gctatactaa ccccagggtt ggtcaatttc gtgccagcca caccgagacc tggtccagag 300tcgctagccg cgtcgctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca tatgactaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaa 4278427RNAArtificial sequenceModA/B 3' UTR 8cucgagcugg uacugcaugc acgcaaugcu agcugccccu uucccguccu ggguaccccg 60agucuccccc gaccucgggu cccagguaug cucccaccuc caccugcccc acucaccacc 120ucugcuaguu ccagacaccu cccaagcacg cagcaaugca gcucaaaacg cuuagccuag 180ccacaccccc acgggaaaca gcagugauua accuuuagca auaaacgaaa guuuaacuaa 240gcuauacuaa ccccaggguu ggucaauuuc gugccagcca caccgagacc ugguccagag 300ucgcuagccg cgucgcuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca uaugacuaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaa 4279153PRTHomo sapiens 9Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu65 70 75 80Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140Cys Gln Ser Ile Ile Ser Thr Leu Thr145 15010465DNAHomo sapiens 10atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420tggattacct tttgtcaaag catcatctca acactgactt gatga 46511465DNAArtificial sequenceHuman optimized IL-2 11atgtacagaa tgcagctgct gtcttgcatt gctctttctc ttgctcttgt gacaaattct 60gctccaacat cttcttcaac aaagaaaaca cagcttcagc ttgaacacct tcttcttgat 120cttcagatga ttctgaatgg aatcaacaat tacaaaaatc caaaactgac aagaatgctg 180acatttaaat tttacatgcc aaagaaagca acagaactga aacaccttca gtgccttgaa 240gaagaactga aacctctgga agaagtgctg aatctggctc agagcaaaaa ttttcacctg 300agaccaagag atctgatcag caacatcaat gtgattgtgc tggaactgaa aggatctgaa 360acaacattca tgtgtgaata tgctgatgaa acagcaacaa ttgtggaatt tctgaacaga 420tggatcacat tttgccagtc aatcatttca acactgacat gatga 46512465RNAHomo sapiens 12auguacagga ugcaacuccu gucuugcauu gcacuaaguc uugcacuugu cacaaacagu 60gcaccuacuu caaguucuac aaagaaaaca cagcuacaac uggagcauuu acugcuggau 120uuacagauga uuuugaaugg aauuaauaau uacaagaauc ccaaacucac caggaugcuc 180acauuuaagu uuuacaugcc caagaaggcc acagaacuga aacaucuuca gugucuagaa 240gaagaacuca aaccucugga ggaagugcua aauuuagcuc aaagcaaaaa cuuucacuua 300agacccaggg acuuaaucag caauaucaac guaauaguuc uggaacuaaa gggaucugaa 360acaacauuca ugugugaaua ugcugaugag acagcaacca uuguagaauu ucugaacaga 420uggauuaccu uuugucaaag caucaucuca acacugacuu gauga 46513465RNAArtificial sequenceHuman optimized IL-2 13auguacagaa ugcagcugcu gucuugcauu gcucuuucuc uugcucuugu gacaaauucu 60gcuccaacau cuucuucaac aaagaaaaca cagcuucagc uugaacaccu ucuucuugau 120cuucagauga uucugaaugg aaucaacaau uacaaaaauc caaaacugac aagaaugcug 180acauuuaaau uuuacaugcc aaagaaagca acagaacuga aacaccuuca gugccuugaa 240gaagaacuga aaccucugga agaagugcug aaucuggcuc agagcaaaaa uuuucaccug 300agaccaagag aucugaucag caacaucaau gugauugugc uggaacugaa aggaucugaa 360acaacauuca ugugugaaua ugcugaugaa acagcaacaa uuguggaauu ucugaacaga 420uggaucacau uuugccaguc aaucauuuca acacugacau gauga 46514539PRTArtificial sequenceHuman IL-12sc 14Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu1 5 10 15Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg145 150 155 160Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn225 230 235 240Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser305 310 315 320Glu Trp Ala Ser Val Pro Cys Ser Gly Ser Ser Gly Gly Gly Gly Ser 325 330 335Pro Gly Gly Gly Ser Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 340 345 350Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 355 360 365Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys 370 375 380Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser385 390 395 400Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys 405 410 415Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala 420 425 430Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr 435 440 445Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys 450 455 460Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu465 470 475 480Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr 485 490 495Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys 500 505 510Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr 515 520 525Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 530 535151623DNAArtificial sequenceHuman non-optimized IL-12sc 15atgtgtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480gggtcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac 780acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960gaatgggcat ctgtgccctg cagtggctct agcggagggg gaggctctcc tggcggggga 1020tctagcagaa acctccccgt ggccactcca gacccaggaa tgttcccatg ccttcaccac 1080tcccaaaacc tgctgagggc cgtcagcaac atgctccaga aggccagaca aactctagaa 1140ttttaccctt gcacttctga ggaaattgat catgaagata tcacaaaaga taaaaccagc 1200acagtggagg cctgtttacc attggaatta accaagaatg agagttgcct aaattccaga 1260gagacctctt tcataactaa tgggagttgc ctggcctcca gaaagacctc ttttatgatg 1320gccctgtgcc ttagtagtat ttatgaagac ttgaagatgt accaggtgga gttcaagacc 1380atgaatgcaa agcttctgat ggatcctaag aggcagatct ttctagatca aaacatgctg 1440gcagttattg atgagctgat gcaggccctg aatttcaaca gtgagactgt gccacaaaaa 1500tcctcccttg aagaaccgga tttttataaa actaaaatca agctctgcat acttcttcat 1560gctttcagaa ttcgggcagt gactattgat agagtgatga gctatctgaa tgcttcctga 1620tga 1623161623DNAArtificial sequenceHuman optimized IL-12sc 16atgtgtcacc agcagctggt gatctcatgg ttctccctgg tatttctggc atctcctctt 60gtcgcaatct gggaactgaa gaaagacgtg tatgtcgttg agctcgactg gtatccggat 120gcgcctggcg agatggtggt gctgacctgt gacaccccag aggaggatgg gatcacttgg 180acccttgatc aatcctccga agtgctcggg tctggcaaga ctctgaccat acaagtgaaa 240gagtttggcg atgccgggca gtacacttgc cataagggcg gagaagttct gtcccactca 300ctgctgctgc tgcacaagaa agaggacgga atttggagta ccgatatcct gaaagatcag 360aaagagccca agaacaaaac cttcttgcgg tgcgaagcca agaactactc agggagattt 420acttgttggt ggctgacgac gatcagcacc gatctgactt tctccgtgaa atcaagtagg 480ggatcatctg accctcaagg agtcacatgt ggagcggcta ctctgagcgc tgaacgcgta 540agaggggaca ataaggagta cgagtatagc gttgagtgcc aagaggatag cgcatgcccc 600gccgccgaag aatcattgcc cattgaagtg atggtggatg ctgtacacaa gctgaagtat 660gagaactaca caagctcctt cttcatccgt gacatcatca aaccagatcc tcctaagaac 720ctccagctta aacctctgaa gaactctaga caggtggaag tgtcttggga gtatcccgac 780acctggtcta caccacattc ctacttcagt ctcacattct gcgttcaggt acagggcaag 840tccaaaaggg agaagaagga tcgggtcttt acagataaaa caagtgccac cgttatatgc 900cggaagaatg cctctatttc tgtgcgtgcg caggacagat actatagcag ctcttggagt 960gaatgggcca gtgtcccatg ttcagggtca tccggtggtg gcggcagccc cggaggcggt 1020agctccagaa atctccctgt ggctacacct gatccaggca tgtttccctg tttgcaccat 1080agccaaaacc tcctgagagc agtcagcaac atgctccaga aagctagaca aacactggaa 1140ttctacccat gcacctccga ggaaatagat cacgaggata tcactaagga caaaacaagc 1200actgtcgaag catgccttcc cttggaactg acaaagaacg agagttgcct taattcaaga 1260gaaacatctt tcattacaaa cggtagctgc ttggcaagca gaaaaacatc ttttatgatg 1320gccctttgtc tgagcagtat ttatgaggat ctcaaaatgt accaggtgga gtttaagacc 1380atgaatgcca agctgctgat ggacccaaag agacagattt tcctcgatca gaatatgctg 1440gctgtgattg atgaactgat gcaggccttg aatttcaaca gcgaaaccgt tccccagaaa 1500agcagtcttg aagaacctga cttttataag accaagatca aactgtgtat tctcctgcat 1560gcctttagaa tcagagcagt cactatagat agagtgatgt cctacctgaa tgcttcctga 1620tga 1623171623RNAArtificial sequenceHuman non-optimized IL-12sc 17augugucacc agcaguuggu caucucuugg uuuucccugg uuuuucuggc aucuccccuc 60guggccauau gggaacugaa gaaagauguu uaugucguag aauuggauug guauccggau 120gccccuggag aaaugguggu ccucaccugu gacaccccug aagaagaugg uaucaccugg 180accuuggacc agagcaguga ggucuuaggc ucuggcaaaa cccugaccau ccaagucaaa 240gaguuuggag augcuggcca guacaccugu cacaaaggag gcgagguucu aagccauucg 300cuccugcugc uucacaaaaa ggaagaugga auuuggucca cugauauuuu aaaggaccag 360aaagaaccca aaaauaagac cuuucuaaga ugcgaggcca agaauuauuc uggacguuuc 420accugcuggu ggcugacgac aaucaguacu gauuugacau ucagugucaa aagcagcaga 480gggucuucug acccccaagg ggugacgugc ggagcugcua cacucucugc agagagaguc 540agaggggaca acaaggagua ugaguacuca guggagugcc aggaggacag ugccugccca 600gcugcugagg agagucugcc cauugagguc augguggaug ccguucacaa gcucaaguau 660gaaaacuaca ccagcagcuu cuucaucagg gacaucauca aaccugaccc acccaagaac 720uugcagcuga agccauuaaa gaauucucgg cagguggagg ucagcuggga guacccugac 780accuggagua cuccacauuc cuacuucucc cugacauucu gcguucaggu ccagggcaag 840agcaagagag aaaagaaaga uagagucuuc acggacaaga ccucagccac ggucaucugc 900cgcaaaaaug ccagcauuag cgugcgggcc caggaccgcu acuauagcuc aucuuggagc 960gaaugggcau cugugcccug caguggcucu agcggagggg gaggcucucc uggcggggga 1020ucuagcagaa accuccccgu ggccacucca gacccaggaa uguucccaug ccuucaccac 1080ucccaaaacc ugcugagggc cgucagcaac augcuccaga aggccagaca aacucuagaa 1140uuuuacccuu gcacuucuga ggaaauugau caugaagaua ucacaaaaga uaaaaccagc 1200acaguggagg ccuguuuacc auuggaauua accaagaaug agaguugccu aaauuccaga 1260gagaccucuu ucauaacuaa ugggaguugc cuggccucca gaaagaccuc uuuuaugaug 1320gcccugugcc uuaguaguau uuaugaagac uugaagaugu accaggugga guucaagacc 1380augaaugcaa agcuucugau ggauccuaag aggcagaucu uucuagauca aaacaugcug 1440gcaguuauug augagcugau gcaggcccug aauuucaaca gugagacugu gccacaaaaa 1500uccucccuug aagaaccgga uuuuuauaaa acuaaaauca agcucugcau acuucuucau 1560gcuuucagaa uucgggcagu gacuauugau agagugauga gcuaucugaa ugcuuccuga 1620uga 1623181623RNAArtificial sequenceHuman optimized IL-12sc 18augugucacc agcagcuggu gaucucaugg uucucccugg uauuucuggc aucuccucuu 60gucgcaaucu gggaacugaa gaaagacgug uaugucguug agcucgacug guauccggau 120gcgccuggcg agaugguggu gcugaccugu gacaccccag aggaggaugg gaucacuugg 180acccuugauc aauccuccga agugcucggg ucuggcaaga cucugaccau acaagugaaa 240gaguuuggcg augccgggca guacacuugc cauaagggcg gagaaguucu gucccacuca 300cugcugcugc ugcacaagaa agaggacgga auuuggagua ccgauauccu gaaagaucag 360aaagagccca agaacaaaac cuucuugcgg ugcgaagcca agaacuacuc agggagauuu 420acuuguuggu ggcugacgac gaucagcacc gaucugacuu ucuccgugaa aucaaguagg 480ggaucaucug acccucaagg agucacaugu ggagcggcua cucugagcgc ugaacgcgua 540agaggggaca auaaggagua cgaguauagc guugagugcc aagaggauag cgcaugcccc 600gccgccgaag aaucauugcc cauugaagug augguggaug cuguacacaa gcugaaguau 660gagaacuaca caagcuccuu cuucauccgu gacaucauca aaccagaucc uccuaagaac 720cuccagcuua aaccucugaa gaacucuaga cagguggaag ugucuuggga guaucccgac 780accuggucua caccacauuc cuacuucagu cucacauucu gcguucaggu acagggcaag 840uccaaaaggg agaagaagga ucgggucuuu acagauaaaa caagugccac cguuauaugc 900cggaagaaug ccucuauuuc ugugcgugcg caggacagau acuauagcag cucuuggagu 960gaaugggcca gugucccaug uucaggguca uccgguggug gcggcagccc cggaggcggu 1020agcuccagaa aucucccugu ggcuacaccu gauccaggca uguuucccug uuugcaccau 1080agccaaaacc uccugagagc agucagcaac augcuccaga aagcuagaca aacacuggaa 1140uucuacccau gcaccuccga ggaaauagau cacgaggaua ucacuaagga caaaacaagc 1200acugucgaag caugccuucc cuuggaacug acaaagaacg agaguugccu uaauucaaga 1260gaaacaucuu ucauuacaaa cgguagcugc uuggcaagca gaaaaacauc uuuuaugaug 1320gcccuuuguc ugagcaguau uuaugaggau cucaaaaugu accaggugga guuuaagacc 1380augaaugcca agcugcugau ggacccaaag agacagauuu uccucgauca gaauaugcug 1440gcugugauug augaacugau gcaggccuug aauuucaaca gcgaaaccgu uccccagaaa 1500agcagucuug aagaaccuga cuuuuauaag accaagauca aacuguguau ucuccugcau 1560gccuuuagaa ucagagcagu cacuauagau agagugaugu ccuaccugaa ugcuuccuga 1620uga

162319188PRTHomo sapiens 19Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25 30Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser 35 40 45Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu 50 55 60Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His65 70 75 80Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100 105 110Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val 115 120 125Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135 140Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro145 150 155 160Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 165 170 175Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu 180 18520570DNAHomo sapiens 20atggccttga cctttgcttt actggtggcc ctcctggtgc tcagctgcaa gtcaagctgc 60tctgtgggct gtgatctgcc tcaaacccac agcctgggta gcaggaggac cttgatgctc 120ctggcacaga tgaggagaat ctctcttttc tcctgcttga aggacagaca tgactttgga 180tttccccagg aggagtttgg caaccagttc caaaaggctg aaaccatccc tgtcctccat 240gagatgatcc agcagatctt caaccttttc agcacaaagg actcatctgc tgcttgggat 300gagaccctcc tagacaaatt ctacactgaa ctctaccagc agctgaatga cctggaagcc 360tgtgtgatac agggggtggg ggtgacagag actcccctga tgaaggagga ctccattctg 420gctgtgagga aatacttcca aagaatcact ctctatctga aagagaagaa atacagccct 480tgtgcctggg aggttgtcag agcagaaatc atgagatctt tttctttgtc aacaaacttg 540caagaaagtt taagaagtaa ggaatgatga 57021570DNAArtificial sequenceHuman optimized IFN-alpha-2b 21atggccctga cttttgccct tctcgtggct ttgttggtgc tgagttgcaa atcttcctgt 60agtgtcggat gtgatctgcc tcaaacccac agtctgggat ctaggagaac actgatgctg 120ttggcacaga tgaggagaat tagcctcttt tcctgcctga aggatagaca tgacttcggc 180tttccccaag aggagtttgg caatcagttc cagaaagcgg aaacgattcc cgttctgcac 240gagatgatcc agcagatctt caacctcttt tcaaccaaag acagctcagc agcctgggat 300gagacactgc tggacaaatt ctacacagaa ctgtatcagc agcttaacga tctggaggca 360tgcgtgatcc aaggggttgg tgtgactgaa actccgctta tgaaggagga ctccattctg 420gctgtacgga agtacttcca gagaataacc ctctatctga aggagaagaa gtactcacca 480tgtgcttggg aagtcgtgag agccgaaatc atgagatcct tcagccttag caccaatctc 540caggaatctc tgagaagcaa agagtgatga 57022570RNAHomo sapiens 22auggccuuga ccuuugcuuu acugguggcc cuccuggugc ucagcugcaa gucaagcugc 60ucugugggcu gugaucugcc ucaaacccac agccugggua gcaggaggac cuugaugcuc 120cuggcacaga ugaggagaau cucucuuuuc uccugcuuga aggacagaca ugacuuugga 180uuuccccagg aggaguuugg caaccaguuc caaaaggcug aaaccauccc uguccuccau 240gagaugaucc agcagaucuu caaccuuuuc agcacaaagg acucaucugc ugcuugggau 300gagacccucc uagacaaauu cuacacugaa cucuaccagc agcugaauga ccuggaagcc 360ugugugauac aggggguggg ggugacagag acuccccuga ugaaggagga cuccauucug 420gcugugagga aauacuucca aagaaucacu cucuaucuga aagagaagaa auacagcccu 480ugugccuggg agguugucag agcagaaauc augagaucuu uuucuuuguc aacaaacuug 540caagaaaguu uaagaaguaa ggaaugauga 57023570RNAArtificial sequenceHuman optimized IFN-alpha-2b 23auggcccuga cuuuugcccu ucucguggcu uuguuggugc ugaguugcaa aucuuccugu 60agugucggau gugaucugcc ucaaacccac agucugggau cuaggagaac acugaugcug 120uuggcacaga ugaggagaau uagccucuuu uccugccuga aggauagaca ugacuucggc 180uuuccccaag aggaguuugg caaucaguuc cagaaagcgg aaacgauucc cguucugcac 240gagaugaucc agcagaucuu caaccucuuu ucaaccaaag acagcucagc agccugggau 300gagacacugc uggacaaauu cuacacagaa cuguaucagc agcuuaacga ucuggaggca 360ugcgugaucc aagggguugg ugugacugaa acuccgcuua ugaaggagga cuccauucug 420gcuguacgga aguacuucca gagaauaacc cucuaucuga aggagaagaa guacucacca 480ugugcuuggg aagucgugag agccgaaauc augagauccu ucagccuuag caccaaucuc 540caggaaucuc ugagaagcaa agagugauga 57024241PRTArtificial sequenceHuman IL-15 sushi 24Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala1 5 10 15Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr 20 25 30Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 35 40 45Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 50 55 60Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala65 70 75 80Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 85 90 95Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Gly Gly Gly Ser Gly 100 105 110Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 115 120 125Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 130 135 140Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro145 150 155 160Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 165 170 175Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 180 185 190Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 195 200 205Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 210 215 220Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr225 230 235 240Ser25729DNAArtificial sequenceHuman IL-15 sushi 25atggccccgc ggcgggcgcg cggctgccgg accctcggtc tcccggcgct gctactgctg 60ctgctgctcc ggccgccggc gacgcggggc atcacgtgcc ctccccccat gtccgtggaa 120cacgcagaca tctgggtcaa gagctacagc ttgtactcca gggagcggta catttgtaac 180tctggtttca agcgtaaagc cggcacgtcc agcctgacgg agtgcgtgtt gaacaaggcc 240acgaatgtcg cccactggac aacccccagt ctcaaatgca ttagagaccc tgccctggtt 300caccaaaggc cagcgccacc cgggggagga tctggcggcg gtgggtctgg cgggggatct 360ggcggaggag gaagcttaca gaactgggtg aatgtaataa gtgatttgaa aaaaattgaa 420gatcttattc aatctatgca tattgatgct actttatata cggaaagtga tgttcacccc 480agttgcaaag taacagcaat gaagtgcttt ctcttggagt tacaagttat ttcacttgag 540tccggagatg caagtattca tgatacagta gaaaatctga tcatcctagc aaacaacagt 600ttgtcttcta atgggaatgt aacagaatct ggatgcaaag aatgtgagga actggaggaa 660aaaaatatta aagaattttt gcagagtttt gtacatattg tccaaatgtt catcaacact 720tcttgatga 72926729RNAArtificial sequenceHuman IL-15 sushi 26auggccccgc ggcgggcgcg cggcugccgg acccucgguc ucccggcgcu gcuacugcug 60cugcugcucc ggccgccggc gacgcggggc aucacgugcc cuccccccau guccguggaa 120cacgcagaca ucugggucaa gagcuacagc uuguacucca gggagcggua cauuuguaac 180ucugguuuca agcguaaagc cggcacgucc agccugacgg agugcguguu gaacaaggcc 240acgaaugucg cccacuggac aacccccagu cucaaaugca uuagagaccc ugcccugguu 300caccaaaggc cagcgccacc cgggggagga ucuggcggcg gugggucugg cgggggaucu 360ggcggaggag gaagcuuaca gaacugggug aauguaauaa gugauuugaa aaaaauugaa 420gaucuuauuc aaucuaugca uauugaugcu acuuuauaua cggaaaguga uguucacccc 480aguugcaaag uaacagcaau gaagugcuuu cucuuggagu uacaaguuau uucacuugag 540uccggagaug caaguauuca ugauacagua gaaaaucuga ucauccuagc aaacaacagu 600uugucuucua augggaaugu aacagaaucu ggaugcaaag aaugugagga acuggaggaa 660aaaaauauua aagaauuuuu gcagaguuuu guacauauug uccaaauguu caucaacacu 720ucuugauga 72927144PRTHomo sapiens 27Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile1 5 10 15Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys65 70 75 80Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 14028438DNAHomo sapiens 28atgtggctcc agagcctgct gctcttgggc actgtggcct gctccatctc tgcacccgcc 60cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120cgtctgctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt 360gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420ccagtccagg agtgatga 43829438RNAHomo sapiens 29auguggcucc agagccugcu gcucuugggc acuguggccu gcuccaucuc ugcacccgcc 60cgcucgccca gccccagcac gcagcccugg gagcauguga augccaucca ggaggcccgg 120cgucugcuga accugaguag agacacugcu gcugagauga augaaacagu agaagucauc 180ucagaaaugu uugaccucca ggagccgacc ugccuacaga cccgccugga gcuguacaag 240cagggccugc ggggcagccu caccaagcuc aagggccccu ugaccaugau ggccagccac 300uacaagcagc acugcccucc aaccccggaa acuuccugug caacccagau uaucaccuuu 360gaaaguuuca aagagaaccu gaaggacuuu cugcuuguca uccccuuuga cugcugggag 420ccaguccagg agugauga 43830161PRTArtificial sequenceModA IL-2 (human IL-2 in combination with a mouse optimized secretion sequence) 30Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala1 5 10 15Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser Ala Pro Thr Ser 20 25 30Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp 35 40 45Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 50 55 60Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu65 70 75 80Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 85 90 95Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 100 105 110Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 115 120 125Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 130 135 140Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu145 150 155 160Thr31959DNAArtificial sequenceModA IL-2 (5'UTR-CDS-3'UTR) 31gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgagagt 60gaccgccccc agaaccctga tcctgctgct gtctggcgcc ctggccctga cagagacatg 120ggccggaagc ggatccgcac ctacttcaag ttctacaaag aaaacacagc tacaactgga 180gcatttactt ctggatttac agatgatttt gaatggaatt aataattaca agaatcccaa 240actcaccagg atgctcacat ttaagtttta catgcccaag aaggccacag aactgaaaca 300tcttcagtgt ctagaagaag aactcaaacc tctggaggaa gtgctaaatt tagctcaaag 360caaaaacttt cacttaagac ccagggactt aatcagcaat atcaacgtaa tagttctgga 420actaaaggga tctgaaacaa cattcatgtg tgaatatgct gatgagacag caaccattgt 480agaatttctg aacagatgga ttaccttttg tcaaagcatc atctcaacac tgacttgact 540cgagagctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc cctaagtcca 600actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc ctaataaaaa 660acatttattt tcattgctgc gtcgagagct cgctttcttg ctgtccaatt tctattaaag 720gttcctttgt tccctaagtc caactactaa actgggggat attatgaagg gccttgagca 780tctggattct gcctaataaa aaacatttat tttcattgct gcgtcgagac ctggtccaga 840gtcgctagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag catatgacta aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 95932959RNAArtificial sequenceModA IL-2 32gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60gaccgccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120ggccggaagc ggauccgcac cuacuucaag uucuacaaag aaaacacagc uacaacugga 180gcauuuacuu cuggauuuac agaugauuuu gaauggaauu aauaauuaca agaaucccaa 240acucaccagg augcucacau uuaaguuuua caugcccaag aaggccacag aacugaaaca 300ucuucagugu cuagaagaag aacucaaacc ucuggaggaa gugcuaaauu uagcucaaag 360caaaaacuuu cacuuaagac ccagggacuu aaucagcaau aucaacguaa uaguucugga 420acuaaaggga ucugaaacaa cauucaugug ugaauaugcu gaugagacag caaccauugu 480agaauuucug aacagaugga uuaccuuuug ucaaagcauc aucucaacac ugacuugacu 540cgagagcucg cuuucuugcu guccaauuuc uauuaaaggu uccuuuguuc ccuaagucca 600acuacuaaac ugggggauau uaugaagggc cuugagcauc uggauucugc cuaauaaaaa 660acauuuauuu ucauugcugc gucgagagcu cgcuuucuug cuguccaauu ucuauuaaag 720guuccuuugu ucccuaaguc caacuacuaa acugggggau auuaugaagg gccuugagca 780ucuggauucu gccuaauaaa aaacauuuau uuucauugcu gcgucgagac cugguccaga 840gucgcuagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag cauaugacua aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 95933161PRTArtificial sequenceModB IL-2 33Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Ala Pro Thr Ser 20 25 30Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp 35 40 45Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 50 55 60Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu65 70 75 80Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 85 90 95Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 100 105 110Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 115 120 125Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 130 135 140Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu145 150 155 160Thr341039DNAArtificial sequenceModB IL-2 (5'UTR-CDS-3'UTR) 34ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatccg cacctacttc 240aagttctaca aagaaaacac agctacaact ggagcattta cttctggatt tacagatgat 300tttgaatgga attaataatt acaagaatcc caaactcacc aggatgctca catttaagtt 360ttacatgccc aagaaggcca cagaactgaa acatcttcag tgtctagaag aagaactcaa 420acctctggag gaagtgctaa atttagctca aagcaaaaac tttcacttaa gacccaggga 480cttaatcagc aatatcaacg taatagttct ggaactaaag ggatctgaaa caacattcat 540gtgtgaatat gctgatgaga cagcaaccat tgtagaattt ctgaacagat ggattacctt 600ttgtcaaagc atcatctcaa cactgacttg actcgacgtc ctggtactgc atgcacgcaa 660tgctagctgc ccctttcccg tcctgggtac cccgagtctc ccccgacctc gggtcccagg 720tatgctccca cctccacctg ccccactcac cacctctgct agttccagac acctcccaag 780cacgcagcaa tgcagctcaa aacgcttagc ctagccacac ccccacggga aacagcagtg 840attaaccttt agcaataaac gaaagtttaa ctaagctata ctaaccccag ggttggtcaa 900tttcgtgcca gccacaccct cgagctagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 960catatgacta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaa 1039351039RNAArtificial sequenceModB IL-2 35ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggauccg caccuacuuc 240aaguucuaca aagaaaacac agcuacaacu ggagcauuua cuucuggauu uacagaugau 300uuugaaugga auuaauaauu acaagaaucc caaacucacc aggaugcuca cauuuaaguu 360uuacaugccc aagaaggcca cagaacugaa acaucuucag ugucuagaag aagaacucaa 420accucuggag gaagugcuaa auuuagcuca aagcaaaaac uuucacuuaa gacccaggga 480cuuaaucagc aauaucaacg uaauaguucu ggaacuaaag ggaucugaaa caacauucau 540gugugaauau gcugaugaga cagcaaccau uguagaauuu cugaacagau ggauuaccuu 600uugucaaagc aucaucucaa cacugacuug acucgacguc cugguacugc augcacgcaa 660ugcuagcugc cccuuucccg uccuggguac cccgagucuc ccccgaccuc gggucccagg 720uaugcuccca ccuccaccug ccccacucac caccucugcu aguuccagac accucccaag 780cacgcagcaa ugcagcucaa aacgcuuagc cuagccacac ccccacggga aacagcagug 840auuaaccuuu agcaauaaac gaaaguuuaa cuaagcuaua cuaaccccag gguuggucaa 900uuucgugcca gccacacccu cgagcuagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 960cauaugacua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020aaaaaaaaaa aaaaaaaaa 103936544PRTArtificial sequenceModA murine IL-12 36Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala1 5 10 15Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser Met Trp Glu Leu 20 25 30Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr Pro Asp Ala Pro 35 40 45Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile 50 55 60Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly Ser Gly Lys Thr65 70 75 80Leu Thr Ile Thr Val Lys Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys 85 90 95His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu Leu Leu His Lys 100 105 110Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn 115 120 125Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr 130 135 140Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys Phe Asn Ile Lys145 150 155 160Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr Cys Gly Met Ala 165 170 175Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys 180 185 190Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro Thr Ala Glu Glu 195 200 205Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr 210 215 220Glu Asn Tyr Ser Thr Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp225 230 235 240Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn Ser Gln Val Glu 245 250 255Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro His Ser Tyr Phe 260 265 270Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys Glu Lys Met Lys 275 280 285Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe Leu Val Glu Lys 290 295 300Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn Val Cys Val Gln Ala305 310 315 320Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp Ala Cys Val Pro 325 330 335Cys Arg Val Arg Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Arg 340 345 350Val Ile Pro Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn 355 360 365Leu Leu Lys Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu 370 375 380Lys His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr385 390 395 400Arg Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His 405 410 415Lys Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg 420 425 430Gly Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys 435 440 445Leu Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln 450 455 460Ala Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu465 470 475 480Asp Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn 485 490 495His Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp 500 505 510Pro Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser 515 520 525Thr Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala 530 535 540372111DNAArtificial sequenceModA murine IL-12 (5'UTR-CDS-3'UTR) 37gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgagagt 60gaccgccccc agaaccctga tcctgctgct gtctggcgcc ctggccctga cagagacatg 120ggccggaagc ggatccatgt gggagctgga gaaagacgtt tatgttgtag aggtggactg 180gactcccgat gcccctggag aaacagtgaa cctcacctgt gacacgcctg aagaagatga 240catcacctgg acctcagacc agagacatgg agtcataggc tctggaaaga ccctgaccat 300cactgtcaaa gagtttctag atgctggcca gtacacctgc cacaaaggag gcgagactct 360gagccactca catctgctgc tccacaagaa ggaaaatgga atttggtcca ctgaaatttt 420aaaaaatttc aaaaacaaga ctttcctgaa gtgtgaagca ccaaattact ccggacggtt 480cacgtgctca tggctggtgc aaagaaacat ggacttgaag ttcaacatca agagcagtag 540cagttcccct gactctcggg cagtgacatg tggaatggcg tctctgtctg cagagaaggt 600cacactggac caaagggact atgagaagta ttcagtgtcc tgccaggagg atgtcacctg 660cccaactgcc gaggagaccc tgcccattga actggcgttg gaagcacggc agcagaataa 720atatgagaac tacagcacca gcttcttcat cagggacatc atcaaaccag acccgcccaa 780gaacttgcag atgaagcctt tgaagaactc acaggtggag gtcagctggg agtaccctga 840ctcctggagc actccccatt cctacttctc cctcaagttc tttgttcgaa tccagcgcaa 900gaaagaaaag atgaaggaga cagaggaggg gtgtaaccag aaaggtgcgt tcctcgtaga 960gaagacatct accgaagtcc aatgcaaagg cgggaatgtc tgcgtgcaag ctcaggatcg 1020ctattacaat tcctcatgca gcaagtgggc atgtgttccc tgcagagtcc gatcggttcc 1080tggagtaggg gtacctggag tgggcagggt cataccggtc tctggacctg ccaggtgtct 1140tagccagtcc cgaaacctgc tgaagaccac agatgacatg gtgaagacgg ccagagaaaa 1200gctgaaacat tattcctgca ctgctgaaga catcgatcat gaagacatca cacgggacca 1260aaccagcaca ttgaagacct gtttaccact ggaactacac aagaacgaga gttgcctggc 1320tactagagag acttcttcca caacaagagg gagctgcctg cccccacaga agacgtcttt 1380gatgatgacc ctgtgccttg gtagcatcta tgaggacttg aagatgtacc agacagagtt 1440ccaggccatc aacgcagcac ttcagaatca caaccatcag cagatcattc tagacaaggg 1500catgctggtg gccatcgatg agctgatgca gtctctgaat cataatggcg agactctgcg 1560ccagaaacct cctgtgggag aagcagaccc ttacagagtg aaaatgaagc tctgcatcct 1620gcttcacgcc ttcagcaccc gcgtcgtgac catcaacagg gtgatgggct atctgtccag 1680cgcctaatag ctcgagagct cgctttcttg ctgtccaatt tctattaaag gttcctttgt 1740tccctaagtc caactactaa actgggggat attatgaagg gccttgagca tctggattct 1800gcctaataaa aaacatttat tttcattgct gcgtcgagag ctcgctttct tgctgtccaa 1860tttctattaa aggttccttt gttccctaag tccaactact aaactggggg atattatgaa 1920gggccttgag catctggatt ctgcctaata aaaaacattt attttcattg ctgcgtcgag 1980acctggtcca gagtcgctag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcatatgac 2040taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa a 2111382111RNAArtificial sequenceModA murine IL-12 38gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60gaccgccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120ggccggaagc ggauccaugu gggagcugga gaaagacguu uauguuguag agguggacug 180gacucccgau gccccuggag aaacagugaa ccucaccugu gacacgccug aagaagauga 240caucaccugg accucagacc agagacaugg agucauaggc ucuggaaaga cccugaccau 300cacugucaaa gaguuucuag augcuggcca guacaccugc cacaaaggag gcgagacucu 360gagccacuca caucugcugc uccacaagaa ggaaaaugga auuuggucca cugaaauuuu 420aaaaaauuuc aaaaacaaga cuuuccugaa gugugaagca ccaaauuacu ccggacgguu 480cacgugcuca uggcuggugc aaagaaacau ggacuugaag uucaacauca agagcaguag 540caguuccccu gacucucggg cagugacaug uggaauggcg ucucugucug cagagaaggu 600cacacuggac caaagggacu augagaagua uucagugucc ugccaggagg augucaccug 660cccaacugcc gaggagaccc ugcccauuga acuggcguug gaagcacggc agcagaauaa 720auaugagaac uacagcacca gcuucuucau cagggacauc aucaaaccag acccgcccaa 780gaacuugcag augaagccuu ugaagaacuc acagguggag gucagcuggg aguacccuga 840cuccuggagc acuccccauu ccuacuucuc ccucaaguuc uuuguucgaa uccagcgcaa 900gaaagaaaag augaaggaga cagaggaggg guguaaccag aaaggugcgu uccucguaga 960gaagacaucu accgaagucc aaugcaaagg cgggaauguc ugcgugcaag cucaggaucg 1020cuauuacaau uccucaugca gcaagugggc auguguuccc ugcagagucc gaucgguucc 1080uggaguaggg guaccuggag ugggcagggu cauaccgguc ucuggaccug ccaggugucu 1140uagccagucc cgaaaccugc ugaagaccac agaugacaug gugaagacgg ccagagaaaa 1200gcugaaacau uauuccugca cugcugaaga caucgaucau gaagacauca cacgggacca 1260aaccagcaca uugaagaccu guuuaccacu ggaacuacac aagaacgaga guugccuggc 1320uacuagagag acuucuucca caacaagagg gagcugccug cccccacaga agacgucuuu 1380gaugaugacc cugugccuug guagcaucua ugaggacuug aagauguacc agacagaguu 1440ccaggccauc aacgcagcac uucagaauca caaccaucag cagaucauuc uagacaaggg 1500caugcuggug gccaucgaug agcugaugca gucucugaau cauaauggcg agacucugcg 1560ccagaaaccu ccugugggag aagcagaccc uuacagagug aaaaugaagc ucugcauccu 1620gcuucacgcc uucagcaccc gcgucgugac caucaacagg gugaugggcu aucuguccag 1680cgccuaauag cucgagagcu cgcuuucuug cuguccaauu ucuauuaaag guuccuuugu 1740ucccuaaguc caacuacuaa acugggggau auuaugaagg gccuugagca ucuggauucu 1800gccuaauaaa aaacauuuau uuucauugcu gcgucgagag cucgcuuucu ugcuguccaa 1860uuucuauuaa agguuccuuu guucccuaag uccaacuacu aaacuggggg auauuaugaa 1920gggccuugag caucuggauu cugccuaaua aaaaacauuu auuuucauug cugcgucgag 1980accuggucca gagucgcuag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac 2040uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa a 211139544PRTArtificial sequenceModB murine IL-12 39Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Met Trp Glu Leu 20 25 30Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr Pro Asp Ala Pro 35 40 45Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile 50 55 60Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly Ser Gly Lys Thr65 70 75 80Leu Thr Ile Thr Val Lys Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys 85 90 95His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu Leu Leu His Lys 100 105 110Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn 115 120 125Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr 130 135 140Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys Phe Asn Ile Lys145 150 155 160Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr Cys Gly Met Ala 165 170 175Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys 180 185 190Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro Thr Ala Glu Glu 195 200 205Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr 210 215 220Glu Asn Tyr Ser Thr Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp225 230 235 240Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn Ser Gln Val Glu 245 250 255Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro His Ser Tyr Phe 260 265 270Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys Glu Lys Met Lys 275 280 285Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe Leu Val Glu Lys 290 295 300Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn Val Cys Val Gln Ala305 310 315 320Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp Ala Cys Val Pro 325 330 335Cys Arg Val Arg Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Arg 340 345 350Val Ile Pro Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn 355 360 365Leu Leu Lys Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu 370 375 380Lys His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr385 390 395 400Arg Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His 405 410 415Lys Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg 420 425 430Gly Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys 435 440 445Leu Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln 450 455 460Ala Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu465 470 475 480Asp Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn 485 490 495His Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp 500 505 510Pro Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser 515 520 525Thr Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala 530 535 540402191DNAArtificial sequenceModB murine IL-12 (5'UTR-CDS-3'UTR) 40ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatcca tgtgggagct 240ggagaaagac gtttatgttg tagaggtgga ctggactccc gatgcccctg gagaaacagt 300gaacctcacc tgtgacacgc ctgaagaaga tgacatcacc tggacctcag accagagaca 360tggagtcata ggctctggaa agaccctgac catcactgtc aaagagtttc tagatgctgg 420ccagtacacc tgccacaaag gaggcgagac tctgagccac tcacatctgc tgctccacaa 480gaaggaaaat ggaatttggt ccactgaaat tttaaaaaat ttcaaaaaca agactttcct 540gaagtgtgaa gcaccaaatt actccggacg gttcacgtgc tcatggctgg tgcaaagaaa 600catggacttg aagttcaaca tcaagagcag tagcagttcc cctgactctc gggcagtgac 660atgtggaatg gcgtctctgt ctgcagagaa ggtcacactg gaccaaaggg actatgagaa 720gtattcagtg tcctgccagg aggatgtcac ctgcccaact gccgaggaga ccctgcccat 780tgaactggcg ttggaagcac ggcagcagaa taaatatgag aactacagca ccagcttctt 840catcagggac atcatcaaac cagacccgcc caagaacttg cagatgaagc ctttgaagaa 900ctcacaggtg gaggtcagct gggagtaccc tgactcctgg agcactcccc attcctactt 960ctccctcaag ttctttgttc gaatccagcg caagaaagaa aagatgaagg agacagagga 1020ggggtgtaac cagaaaggtg cgttcctcgt agagaagaca tctaccgaag tccaatgcaa 1080aggcgggaat gtctgcgtgc aagctcagga tcgctattac aattcctcat gcagcaagtg 1140ggcatgtgtt ccctgcagag tccgatcggt tcctggagta ggggtacctg gagtgggcag 1200ggtcataccg gtctctggac ctgccaggtg tcttagccag tcccgaaacc tgctgaagac 1260cacagatgac atggtgaaga cggccagaga aaagctgaaa cattattcct gcactgctga 1320agacatcgat catgaagaca tcacacggga ccaaaccagc acattgaaga cctgtttacc 1380actggaacta cacaagaacg agagttgcct ggctactaga gagacttctt ccacaacaag 1440agggagctgc ctgcccccac agaagacgtc tttgatgatg accctgtgcc ttggtagcat 1500ctatgaggac ttgaagatgt accagacaga gttccaggcc atcaacgcag cacttcagaa 1560tcacaaccat cagcagatca ttctagacaa gggcatgctg gtggccatcg atgagctgat 1620gcagtctctg aatcataatg gcgagactct gcgccagaaa cctcctgtgg gagaagcaga 1680cccttacaga gtgaaaatga agctctgcat cctgcttcac gccttcagca cccgcgtcgt 1740gaccatcaac agggtgatgg gctatctgtc cagcgcctaa tagctcgacg tcctggtact 1800gcatgcacgc aatgctagct gcccctttcc cgtcctgggt accccgagtc tcccccgacc 1860tcgggtccca ggtatgctcc cacctccacc tgccccactc accacctctg ctagttccag 1920acacctccca agcacgcagc aatgcagctc aaaacgctta gcctagccac acccccacgg 1980gaaacagcag tgattaacct ttagcaataa acgaaagttt aactaagcta tactaacccc 2040agggttggtc aatttcgtgc cagccacacc ctcgagctag caaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa agcatatgac taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2191412191RNAArtificial sequenceModB murine IL-12 41ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggaucca ugugggagcu 240ggagaaagac guuuauguug uagaggugga cuggacuccc gaugccccug gagaaacagu 300gaaccucacc ugugacacgc cugaagaaga ugacaucacc uggaccucag accagagaca 360uggagucaua ggcucuggaa agacccugac caucacuguc aaagaguuuc uagaugcugg 420ccaguacacc ugccacaaag gaggcgagac ucugagccac ucacaucugc ugcuccacaa 480gaaggaaaau ggaauuuggu ccacugaaau uuuaaaaaau uucaaaaaca agacuuuccu 540gaagugugaa gcaccaaauu acuccggacg guucacgugc ucauggcugg ugcaaagaaa 600cauggacuug aaguucaaca ucaagagcag uagcaguucc ccugacucuc gggcagugac 660auguggaaug gcgucucugu cugcagagaa ggucacacug gaccaaaggg acuaugagaa 720guauucagug uccugccagg aggaugucac cugcccaacu gccgaggaga cccugcccau 780ugaacuggcg uuggaagcac ggcagcagaa uaaauaugag aacuacagca ccagcuucuu 840caucagggac aucaucaaac cagacccgcc caagaacuug cagaugaagc cuuugaagaa 900cucacaggug gaggucagcu gggaguaccc ugacuccugg agcacucccc auuccuacuu 960cucccucaag uucuuuguuc gaauccagcg caagaaagaa aagaugaagg agacagagga 1020gggguguaac cagaaaggug cguuccucgu agagaagaca ucuaccgaag uccaaugcaa 1080aggcgggaau gucugcgugc aagcucagga ucgcuauuac aauuccucau gcagcaagug 1140ggcauguguu cccugcagag uccgaucggu uccuggagua gggguaccug gagugggcag 1200ggucauaccg gucucuggac cugccaggug ucuuagccag ucccgaaacc ugcugaagac 1260cacagaugac auggugaaga cggccagaga aaagcugaaa cauuauuccu gcacugcuga 1320agacaucgau caugaagaca ucacacggga ccaaaccagc acauugaaga ccuguuuacc 1380acuggaacua cacaagaacg agaguugccu ggcuacuaga gagacuucuu ccacaacaag 1440agggagcugc cugcccccac agaagacguc uuugaugaug acccugugcc uugguagcau 1500cuaugaggac uugaagaugu accagacaga guuccaggcc aucaacgcag cacuucagaa 1560ucacaaccau cagcagauca

uucuagacaa gggcaugcug guggccaucg augagcugau 1620gcagucucug aaucauaaug gcgagacucu gcgccagaaa ccuccugugg gagaagcaga 1680cccuuacaga gugaaaauga agcucugcau ccugcuucac gccuucagca cccgcgucgu 1740gaccaucaac agggugaugg gcuaucuguc cagcgccuaa uagcucgacg uccugguacu 1800gcaugcacgc aaugcuagcu gccccuuucc cguccugggu accccgaguc ucccccgacc 1860ucggguccca gguaugcucc caccuccacc ugccccacuc accaccucug cuaguuccag 1920acaccuccca agcacgcagc aaugcagcuc aaaacgcuua gccuagccac acccccacgg 1980gaaacagcag ugauuaaccu uuagcaauaa acgaaaguuu aacuaagcua uacuaacccc 2040aggguugguc aauuucgugc cagccacacc cucgagcuag caaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa agcauaugac uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 219142190PRTArtificial sequenceModA murine IFN-alpha-4 42Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala1 5 10 15Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser Cys Asp Leu Pro 20 25 30His Thr Tyr Asn Leu Gly Asn Lys Arg Ala Leu Thr Val Leu Glu Glu 35 40 45Met Arg Arg Leu Pro Pro Leu Ser Cys Leu Lys Asp Arg Lys Asp Phe 50 55 60Gly Phe Pro Leu Glu Lys Val Asp Asn Gln Gln Ile Gln Lys Ala Gln65 70 75 80Ala Ile Leu Val Leu Arg Asp Leu Thr Gln Gln Ile Leu Asn Leu Phe 85 90 95Thr Ser Lys Asp Leu Ser Ala Thr Trp Asn Ala Thr Leu Leu Asp Ser 100 105 110Phe Cys Asn Asp Leu His Gln Gln Leu Asn Asp Leu Lys Ala Cys Val 115 120 125Met Gln Glu Pro Pro Leu Thr Gln Glu Asp Ser Leu Leu Ala Val Arg 130 135 140Thr Tyr Phe His Arg Ile Thr Val Tyr Leu Arg Lys Lys Lys His Ser145 150 155 160Leu Cys Ala Trp Glu Val Ile Arg Ala Glu Val Trp Arg Ala Leu Ser 165 170 175Ser Ser Thr Asn Leu Leu Ala Arg Leu Ser Glu Glu Lys Glu 180 185 190431049DNAArtificial sequenceModA murine IFN-alpha-4 (5'UTR-CDS-3'UTR) 43gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgagagt 60gaccgccccc agaaccctga tcctgctgct gtctggcgcc ctggccctga cagagacatg 120ggccggaagc ggatcctgtg acctgcctca cacttataac ctcgggaaca agagggcctt 180gacagtcctg gaagaaatga gaagactccc ccctctttcc tgcctgaagg acaggaagga 240ttttggattc cccttggaga aggtggataa ccaacagatc cagaaggctc aagccatcct 300tgtgctaaga gatcttaccc agcagatttt gaacctcttc acatcaaaag acttgtctgc 360tacttggaat gcaactctcc tagactcatt ctgcaatgac ctccatcagc agctcaatga 420tctcaaagcc tgtgtgatgc aggaacctcc tctgacccag gaagactccc tgctggctgt 480gaggacatac ttccacagga tcactgtgta cctgagaaag aagaaacaca gcctctgtgc 540ctgggaggtg atcagagcag aagtctggag agccctctct tcctcaacca acttgctggc 600aagactgagt gaggagaagg agtgataact cgagagctcg ctttcttgct gtccaatttc 660tattaaaggt tcctttgttc cctaagtcca actactaaac tgggggatat tatgaagggc 720cttgagcatc tggattctgc ctaataaaaa acatttattt tcattgctgc gtcgagagct 780cgctttcttg ctgtccaatt tctattaaag gttcctttgt tccctaagtc caactactaa 840actgggggat attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat 900tttcattgct gcgtcgagac ctggtccaga gtcgctagca aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaag catatgacta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1049441049RNAArtificial sequenceModA murine IFN-alpha-4 44gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60gaccgccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120ggccggaagc ggauccugug accugccuca cacuuauaac cucgggaaca agagggccuu 180gacaguccug gaagaaauga gaagacuccc cccucuuucc ugccugaagg acaggaagga 240uuuuggauuc cccuuggaga agguggauaa ccaacagauc cagaaggcuc aagccauccu 300ugugcuaaga gaucuuaccc agcagauuuu gaaccucuuc acaucaaaag acuugucugc 360uacuuggaau gcaacucucc uagacucauu cugcaaugac cuccaucagc agcucaauga 420ucucaaagcc ugugugaugc aggaaccucc ucugacccag gaagacuccc ugcuggcugu 480gaggacauac uuccacagga ucacugugua ccugagaaag aagaaacaca gccucugugc 540cugggaggug aucagagcag aagucuggag agcccucucu uccucaacca acuugcuggc 600aagacugagu gaggagaagg agugauaacu cgagagcucg cuuucuugcu guccaauuuc 660uauuaaaggu uccuuuguuc ccuaagucca acuacuaaac ugggggauau uaugaagggc 720cuugagcauc uggauucugc cuaauaaaaa acauuuauuu ucauugcugc gucgagagcu 780cgcuuucuug cuguccaauu ucuauuaaag guuccuuugu ucccuaaguc caacuacuaa 840acugggggau auuaugaagg gccuugagca ucuggauucu gccuaauaaa aaacauuuau 900uuucauugcu gcgucgagac cugguccaga gucgcuagca aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaag cauaugacua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 104945190PRTArtificial sequenceModB murine IFN-alpha-4 45Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Cys Asp Leu Pro 20 25 30His Thr Tyr Asn Leu Gly Asn Lys Arg Ala Leu Thr Val Leu Glu Glu 35 40 45Met Arg Arg Leu Pro Pro Leu Ser Cys Leu Lys Asp Arg Lys Asp Phe 50 55 60Gly Phe Pro Leu Glu Lys Val Asp Asn Gln Gln Ile Gln Lys Ala Gln65 70 75 80Ala Ile Leu Val Leu Arg Asp Leu Thr Gln Gln Ile Leu Asn Leu Phe 85 90 95Thr Ser Lys Asp Leu Ser Ala Thr Trp Asn Ala Thr Leu Leu Asp Ser 100 105 110Phe Cys Asn Asp Leu His Gln Gln Leu Asn Asp Leu Lys Ala Cys Val 115 120 125Met Gln Glu Pro Pro Leu Thr Gln Glu Asp Ser Leu Leu Ala Val Arg 130 135 140Thr Tyr Phe His Arg Ile Thr Val Tyr Leu Arg Lys Lys Lys His Ser145 150 155 160Leu Cys Ala Trp Glu Val Ile Arg Ala Glu Val Trp Arg Ala Leu Ser 165 170 175Ser Ser Thr Asn Leu Leu Ala Arg Leu Ser Glu Glu Lys Glu 180 185 190461129DNAArtificial sequenceModB murine IFN-alpha-4 (5'UTR-CDS-3'UTR) 46ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatcct gtgacctgcc 240tcacacttat aacctcggga acaagagggc cttgacagtc ctggaagaaa tgagaagact 300cccccctctt tcctgcctga aggacaggaa ggattttgga ttccccttgg agaaggtgga 360taaccaacag atccagaagg ctcaagccat ccttgtgcta agagatctta cccagcagat 420tttgaacctc ttcacatcaa aagacttgtc tgctacttgg aatgcaactc tcctagactc 480attctgcaat gacctccatc agcagctcaa tgatctcaaa gcctgtgtga tgcaggaacc 540tcctctgacc caggaagact ccctgctggc tgtgaggaca tacttccaca ggatcactgt 600gtacctgaga aagaagaaac acagcctctg tgcctgggag gtgatcagag cagaagtctg 660gagagccctc tcttcctcaa ccaacttgct ggcaagactg agtgaggaga aggagtgata 720actcgacgtc ctggtactgc atgcacgcaa tgctagctgc ccctttcccg tcctgggtac 780cccgagtctc ccccgacctc gggtcccagg tatgctccca cctccacctg ccccactcac 840cacctctgct agttccagac acctcccaag cacgcagcaa tgcagctcaa aacgcttagc 900ctagccacac ccccacggga aacagcagtg attaaccttt agcaataaac gaaagtttaa 960ctaagctata ctaaccccag ggttggtcaa tttcgtgcca gccacaccct cgagctagca 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaag catatgacta aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1129471129RNAArtificial sequenceModB murine IFN-alpha-4 47ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggauccu gugaccugcc 240ucacacuuau aaccucggga acaagagggc cuugacaguc cuggaagaaa ugagaagacu 300ccccccucuu uccugccuga aggacaggaa ggauuuugga uuccccuugg agaaggugga 360uaaccaacag auccagaagg cucaagccau ccuugugcua agagaucuua cccagcagau 420uuugaaccuc uucacaucaa aagacuuguc ugcuacuugg aaugcaacuc uccuagacuc 480auucugcaau gaccuccauc agcagcucaa ugaucucaaa gccuguguga ugcaggaacc 540uccucugacc caggaagacu cccugcuggc ugugaggaca uacuuccaca ggaucacugu 600guaccugaga aagaagaaac acagccucug ugccugggag gugaucagag cagaagucug 660gagagcccuc ucuuccucaa ccaacuugcu ggcaagacug agugaggaga aggagugaua 720acucgacguc cugguacugc augcacgcaa ugcuagcugc cccuuucccg uccuggguac 780cccgagucuc ccccgaccuc gggucccagg uaugcuccca ccuccaccug ccccacucac 840caccucugcu aguuccagac accucccaag cacgcagcaa ugcagcucaa aacgcuuagc 900cuagccacac ccccacggga aacagcagug auuaaccuuu agcaauaaac gaaaguuuaa 960cuaagcuaua cuaaccccag gguuggucaa uuucgugcca gccacacccu cgagcuagca 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaag cauaugacua aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 112948232PRTArtificial sequenceModA murine IL-15 sushi 48Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Thr Thr Cys Pro 20 25 30Pro Pro Val Ser Ile Glu His Ala Asp Ile Arg Val Lys Asn Tyr Ser 35 40 45Val Asn Ser Arg Glu Arg Tyr Val Cys Asn Ser Gly Phe Lys Arg Lys 50 55 60Ala Gly Thr Ser Thr Leu Ile Glu Cys Val Ile Asn Lys Asn Thr Asn65 70 75 80Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ser 85 90 95Leu Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 100 105 110Ser Gly Gly Ser Gly Gly Asn Trp Ile Asp Val Arg Tyr Asp Leu Glu 115 120 125Lys Ile Glu Ser Leu Ile Gln Ser Ile His Ile Asp Thr Thr Leu Tyr 130 135 140Thr Asp Ser Asp Phe His Pro Ser Cys Lys Val Thr Ala Met Asn Cys145 150 155 160Phe Leu Leu Glu Leu Gln Val Ile Leu His Glu Tyr Ser Asn Met Thr 165 170 175Leu Asn Glu Thr Val Arg Asn Val Leu Tyr Leu Ala Asn Ser Thr Leu 180 185 190Ser Ser Asn Lys Asn Val Ala Glu Ser Gly Cys Lys Glu Cys Glu Glu 195 200 205Leu Glu Glu Lys Thr Phe Thr Glu Phe Leu Gln Ser Phe Ile Arg Ile 210 215 220Val Gln Met Phe Ile Asn Thr Ser225 230491162DNAArtificial sequenceModA murine IL-15 sushi (5'UTR-CDS-3'UTR) 49gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgggcgc 60catggcccct agaacattgc tcctgctgct ggccgctgcc ctggccccta cacagacaag 120agctggacct ggatccacca cgtgtccacc tcccgtatct attgagcatg ctgacatccg 180ggtcaagaat tacagtgtga actccaggga gaggtatgtc tgtaactctg gctttaagcg 240gaaagctgga acatccaccc tgattgagtg tgtgatcaac aagaacacaa atgttgccca 300ctggacaact cccagcctca agtgcatcag agacccctcc ctagctggag ggagcggagg 360ctctggcgga agcggcgggt ctggaggctc cgggggaagc ggcggaaatt ggatcgacgt 420gcgctacgac ctggaaaaga tcgagagcct gatccagagc atccacatcg acaccaccct 480gtacaccgac agcgacttcc accccagctg caaagtgacc gctatgaact gcttcctgct 540ggaactgcaa gtgatcctgc acgagtacag caacatgacc ctgaacgaga cagtgcggaa 600cgtgctgtac ctggccaaca gcaccctgag cagcaacaag aacgtggccg agagcggctg 660caaagagtgc gaggaactgg aagaaaagac cttcaccgag tttctgcaga gcttcatcag 720gatcgtgcag atgttcatca acacctcttg atgagtcgac gtcctggtac tgcatgcacg 780caatgctagc tgcccctttc ccgtcctggg taccccgagt ctcccccgac ctcgggtccc 840aggtatgctc ccacctccac ctgccccact caccacctct gctagttcca gacacctccc 900aagcacgcag caatgcagct caaaacgctt agcctagcca cacccccacg ggaaacagca 960gtgattaacc tttagcaata aacgaaagtt taactaagct atactaaccc cagggttggt 1020caatttcgtg ccagccacac cctcgagcta gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aagcatatga ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aa 1162501162RNAArtificial sequenceModA murine IL-15 sushi 50gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugggcgc 60cauggccccu agaacauugc uccugcugcu ggccgcugcc cuggccccua cacagacaag 120agcuggaccu ggauccacca cguguccacc ucccguaucu auugagcaug cugacauccg 180ggucaagaau uacaguguga acuccaggga gagguauguc uguaacucug gcuuuaagcg 240gaaagcugga acauccaccc ugauugagug ugugaucaac aagaacacaa auguugccca 300cuggacaacu cccagccuca agugcaucag agaccccucc cuagcuggag ggagcggagg 360cucuggcgga agcggcgggu cuggaggcuc cgggggaagc ggcggaaauu ggaucgacgu 420gcgcuacgac cuggaaaaga ucgagagccu gauccagagc auccacaucg acaccacccu 480guacaccgac agcgacuucc accccagcug caaagugacc gcuaugaacu gcuuccugcu 540ggaacugcaa gugauccugc acgaguacag caacaugacc cugaacgaga cagugcggaa 600cgugcuguac cuggccaaca gcacccugag cagcaacaag aacguggccg agagcggcug 660caaagagugc gaggaacugg aagaaaagac cuucaccgag uuucugcaga gcuucaucag 720gaucgugcag auguucauca acaccucuug augagucgac guccugguac ugcaugcacg 780caaugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc 840agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc 900aagcacgcag caaugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca 960gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu 1020caauuucgug ccagccacac ccucgagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aa 116251232PRTArtificial sequenceModB murine IL-15 sushi 51Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Thr Thr Cys Pro 20 25 30Pro Pro Val Ser Ile Glu His Ala Asp Ile Arg Val Lys Asn Tyr Ser 35 40 45Val Asn Ser Arg Glu Arg Tyr Val Cys Asn Ser Gly Phe Lys Arg Lys 50 55 60Ala Gly Thr Ser Thr Leu Ile Glu Cys Val Ile Asn Lys Asn Thr Asn65 70 75 80Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ser 85 90 95Leu Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 100 105 110Ser Gly Gly Ser Gly Gly Asn Trp Ile Asp Val Arg Tyr Asp Leu Glu 115 120 125Lys Ile Glu Ser Leu Ile Gln Ser Ile His Ile Asp Thr Thr Leu Tyr 130 135 140Thr Asp Ser Asp Phe His Pro Ser Cys Lys Val Thr Ala Met Asn Cys145 150 155 160Phe Leu Leu Glu Leu Gln Val Ile Leu His Glu Tyr Ser Asn Met Thr 165 170 175Leu Asn Glu Thr Val Arg Asn Val Leu Tyr Leu Ala Asn Ser Thr Leu 180 185 190Ser Ser Asn Lys Asn Val Ala Glu Ser Gly Cys Lys Glu Cys Glu Glu 195 200 205Leu Glu Glu Lys Thr Phe Thr Glu Phe Leu Gln Ser Phe Ile Arg Ile 210 215 220Val Gln Met Phe Ile Asn Thr Ser225 230521255DNAArtificial sequenceModB murine IL-15 sushi (5'UTR-CDS-3'UTR) 52ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatcca ccacgtgtcc 240acctcccgta tctattgagc atgctgacat ccgggtcaag aattacagtg tgaactccag 300ggagaggtat gtctgtaact ctggctttaa gcggaaagct ggaacatcca ccctgattga 360gtgtgtgatc aacaagaaca caaatgttgc ccactggaca actcccagcc tcaagtgcat 420cagagacccc tccctagctg gagggagcgg aggctctggc ggaagcggcg ggtctggagg 480ctccggggga agcggcggaa attggatcga cgtgcgctac gacctggaaa agatcgagag 540cctgatccag agcatccaca tcgacaccac cctgtacacc gacagcgact tccaccccag 600ctgcaaagtg accgctatga actgcttcct gctggaactg caagtgatcc tgcacgagta 660cagcaacatg accctgaacg agacagtgcg gaacgtgctg tacctggcca acagcaccct 720gagcagcaac aagaacgtgg ccgagagcgg ctgcaaagag tgcgaggaac tggaagaaaa 780gaccttcacc gagtttctgc agagcttcat caggatcgtg cagatgttca tcaacacctc 840ttgatgagtc gacgtcctgg tactgcatgc acgcaatgct agctgcccct ttcccgtcct 900gggtaccccg agtctccccc gacctcgggt cccaggtatg ctcccacctc cacctgcccc 960actcaccacc tctgctagtt ccagacacct cccaagcacg cagcaatgca gctcaaaacg 1020cttagcctag ccacaccccc acgggaaaca gcagtgatta acctttagca ataaacgaaa 1080gtttaactaa gctatactaa ccccagggtt ggtcaatttc gtgccagcca caccctcgag 1140ctagcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagcata tgactaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1255531255RNAArtificial sequenceModB murine IL-15 sushi 53ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggaucca ccacgugucc 240accucccgua ucuauugagc augcugacau ccgggucaag aauuacagug ugaacuccag 300ggagagguau gucuguaacu cuggcuuuaa gcggaaagcu ggaacaucca cccugauuga 360gugugugauc aacaagaaca caaauguugc ccacuggaca acucccagcc ucaagugcau 420cagagacccc ucccuagcug gagggagcgg aggcucuggc ggaagcggcg ggucuggagg 480cuccggggga agcggcggaa auuggaucga cgugcgcuac gaccuggaaa agaucgagag 540ccugauccag

agcauccaca ucgacaccac ccuguacacc gacagcgacu uccaccccag 600cugcaaagug accgcuauga acugcuuccu gcuggaacug caagugaucc ugcacgagua 660cagcaacaug acccugaacg agacagugcg gaacgugcug uaccuggcca acagcacccu 720gagcagcaac aagaacgugg ccgagagcgg cugcaaagag ugcgaggaac uggaagaaaa 780gaccuucacc gaguuucugc agagcuucau caggaucgug cagauguuca ucaacaccuc 840uugaugaguc gacguccugg uacugcaugc acgcaaugcu agcugccccu uucccguccu 900ggguaccccg agucuccccc gaccucgggu cccagguaug cucccaccuc caccugcccc 960acucaccacc ucugcuaguu ccagacaccu cccaagcacg cagcaaugca gcucaaaacg 1020cuuagccuag ccacaccccc acgggaaaca gcagugauua accuuuagca auaaacgaaa 1080guuuaacuaa gcuauacuaa ccccaggguu ggucaauuuc gugccagcca cacccucgag 1140cuagcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagcaua ugacuaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 125554141PRTArtificial sequenceModA murine GM-CSF 54Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu1 5 10 15Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 25 30Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 35 40 45Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 50 55 60Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu65 70 75 80Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 85 90 95Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 100 105 110Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 115 120 125Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys 130 135 14055908DNAArtificial sequenceModA murine GM-CSF (5'UTR-CDS-3'UTR) 55gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgtggct 60gcagaacctg ctgttcctgg gcatcgtggt gtacagcctg agcgccccca ccaggagccc 120catcaccgtg accaggccct ggaagcacgt ggaggccatc aaggaggccc tgaacctgct 180ggacgacatg cccgtgaccc tgaacgagga ggtggaggtg gtgagcaacg agttcagctt 240caagaagctg acctgcgtgc agaccaggct gaagatcttc gagcagggcc tgaggggcaa 300cttcaccaag ctgaagggcg ccctgaacat gaccgccagc tactaccaga cctactgccc 360ccccaccccc gagaccgact gcgagaccca ggtgaccacc tacgccgact tcatcgacag 420cctgaagacc ttcctgaccg acatcccctt cgagtgcaag aagcccggcc agaagtgatg 480actcgagctg gtactgcatg cacgcaatgc tagctgcccc tttcccgtcc tgggtacccc 540gagtctcccc cgacctcggg tcccaggtat gctcccacct ccacctgccc cactcaccac 600ctctgctagt tccagacacc tcccaagcac gcagcaatgc agctcaaaac gcttagccta 660gccacacccc cacgggaaac agcagtgatt aacctttagc aataaacgaa agtttaacta 720agctatacta accccagggt tggtcaattt cgtgccagcc acaccgagac ctggtccaga 780gtcgctagcc gcgtcgctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagc atatgactaa 840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaa 90856908RNAArtificial sequenceModA murine GM-CSF 56gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguggcu 60gcagaaccug cuguuccugg gcaucguggu guacagccug agcgccccca ccaggagccc 120caucaccgug accaggcccu ggaagcacgu ggaggccauc aaggaggccc ugaaccugcu 180ggacgacaug cccgugaccc ugaacgagga gguggaggug gugagcaacg aguucagcuu 240caagaagcug accugcgugc agaccaggcu gaagaucuuc gagcagggcc ugaggggcaa 300cuucaccaag cugaagggcg cccugaacau gaccgccagc uacuaccaga ccuacugccc 360ccccaccccc gagaccgacu gcgagaccca ggugaccacc uacgccgacu ucaucgacag 420ccugaagacc uuccugaccg acauccccuu cgagugcaag aagcccggcc agaagugaug 480acucgagcug guacugcaug cacgcaaugc uagcugcccc uuucccgucc uggguacccc 540gagucucccc cgaccucggg ucccagguau gcucccaccu ccaccugccc cacucaccac 600cucugcuagu uccagacacc ucccaagcac gcagcaaugc agcucaaaac gcuuagccua 660gccacacccc cacgggaaac agcagugauu aaccuuuagc aauaaacgaa aguuuaacua 720agcuauacua accccagggu uggucaauuu cgugccagcc acaccgagac cugguccaga 780gucgcuagcc gcgucgcuaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagc auaugacuaa 840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaa 90857141PRTArtificial sequenceModB murine GM-CSF 57Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu1 5 10 15Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 25 30Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 35 40 45Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 50 55 60Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu65 70 75 80Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 85 90 95Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 100 105 110Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 115 120 125Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys 130 135 140581001DNAArtificial sequenceModB murine GM-CSF (5'UTR-CDS-3'UTR) 58ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatgtg gctgcagaac ctgctgttcc tgggcatcgt 180ggtgtacagc ctgagcgccc ccaccaggag ccccatcacc gtgaccaggc cctggaagca 240cgtggaggcc atcaaggagg ccctgaacct gctggacgac atgcccgtga ccctgaacga 300ggaggtggag gtggtgagca acgagttcag cttcaagaag ctgacctgcg tgcagaccag 360gctgaagatc ttcgagcagg gcctgagggg caacttcacc aagctgaagg gcgccctgaa 420catgaccgcc agctactacc agacctactg cccccccacc cccgagaccg actgcgagac 480ccaggtgacc acctacgccg acttcatcga cagcctgaag accttcctga ccgacatccc 540cttcgagtgc aagaagcccg gccagaagtg atgactcgag ctggtactgc atgcacgcaa 600tgctagctgc ccctttcccg tcctgggtac cccgagtctc ccccgacctc gggtcccagg 660tatgctccca cctccacctg ccccactcac cacctctgct agttccagac acctcccaag 720cacgcagcaa tgcagctcaa aacgcttagc ctagccacac ccccacggga aacagcagtg 780attaaccttt agcaataaac gaaagtttaa ctaagctata ctaaccccag ggttggtcaa 840tttcgtgcca gccacaccga gacctggtcc agagtcgcta gccgcgtcgc taaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa agcatatgac taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1001591001RNAArtificial sequenceModB murine GM-CSF 59ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccaugug gcugcagaac cugcuguucc ugggcaucgu 180gguguacagc cugagcgccc ccaccaggag ccccaucacc gugaccaggc ccuggaagca 240cguggaggcc aucaaggagg cccugaaccu gcuggacgac augcccguga cccugaacga 300ggagguggag guggugagca acgaguucag cuucaagaag cugaccugcg ugcagaccag 360gcugaagauc uucgagcagg gccugagggg caacuucacc aagcugaagg gcgcccugaa 420caugaccgcc agcuacuacc agaccuacug cccccccacc cccgagaccg acugcgagac 480ccaggugacc accuacgccg acuucaucga cagccugaag accuuccuga ccgacauccc 540cuucgagugc aagaagcccg gccagaagug augacucgag cugguacugc augcacgcaa 600ugcuagcugc cccuuucccg uccuggguac cccgagucuc ccccgaccuc gggucccagg 660uaugcuccca ccuccaccug ccccacucac caccucugcu aguuccagac accucccaag 720cacgcagcaa ugcagcucaa aacgcuuagc cuagccacac ccccacggga aacagcagug 780auuaaccuuu agcaauaaac gaaaguuuaa cuaagcuaua cuaaccccag gguuggucaa 840uuucgugcca gccacaccga gaccuggucc agagucgcua gccgcgucgc uaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 100160187PRTArtificial sequenceModA FLT3L (human FLT3L in combination with a mouse optimized secretion sequence) 60Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Thr Gln Asp Cys 20 25 30Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg 35 40 45Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser 50 55 60Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu65 70 75 80Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met 85 90 95Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys 100 105 110Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn 115 120 125Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys 130 135 140Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys145 150 155 160Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu 165 170 175Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Pro 180 185611027DNAArtificial sequenceModA FLT3L (5'UTR-CDS-3'UTR) 61gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgggcgc 60catggcccct agaacattgc tcctgctgct ggccgctgcc ctggccccta cacagacaag 120agctggacct ggatccaccc aggactgcag cttccagcac tcccctatct cctccgactt 180cgccgtgaag atccgggagc tgtccgatta cctgctgcag gactaccctg tgaccgtggc 240cagcaacctg caggacgaag aactgtgtgg cggcctgtgg cggctggtgc tggcccagcg 300gtggatggaa cggctgaaaa ccgtggccgg ctccaagatg cagggcctgc tcgagcgggt 360gaacaccgag atccacttcg tgaccaagtg cgccttccag cctcctcctt cctgcctgcg 420gttcgtgcag accaacatct cccggctgct gcaggaaacc tccgagcagc tggtcgccct 480gaagccttgg atcacccggc agaacttctc ccggtgtctg gaactccagt gtcagcccga 540ctcctccacc ctgcctcctc cctggtcccc caggcctctg gaagccaccg cccctaccgc 600cccacagcct ccttgatagg tcgacgtcct ggtactgcat gcacgcaatg ctagctgccc 660ctttcccgtc ctgggtaccc cgagtctccc ccgacctcgg gtcccaggta tgctcccacc 720tccacctgcc ccactcacca cctctgctag ttccagacac ctcccaagca cgcagcaatg 780cagctcaaaa cgcttagcct agccacaccc ccacgggaaa cagcagtgat taacctttag 840caataaacga aagtttaact aagctatact aaccccaggg ttggtcaatt tcgtgccagc 900cacaccctcg agctagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca tatgactaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaa 1027621027RNAArtificial sequenceModA FLT3L 62gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugggcgc 60cauggccccu agaacauugc uccugcugcu ggccgcugcc cuggccccua cacagacaag 120agcuggaccu ggauccaccc aggacugcag cuuccagcac uccccuaucu ccuccgacuu 180cgccgugaag auccgggagc uguccgauua ccugcugcag gacuacccug ugaccguggc 240cagcaaccug caggacgaag aacugugugg cggccugugg cggcuggugc uggcccagcg 300guggauggaa cggcugaaaa ccguggccgg cuccaagaug cagggccugc ucgagcgggu 360gaacaccgag auccacuucg ugaccaagug cgccuuccag ccuccuccuu ccugccugcg 420guucgugcag accaacaucu cccggcugcu gcaggaaacc uccgagcagc uggucgcccu 480gaagccuugg aucacccggc agaacuucuc ccggugucug gaacuccagu gucagcccga 540cuccuccacc cugccuccuc ccuggucccc caggccucug gaagccaccg ccccuaccgc 600cccacagccu ccuugauagg ucgacguccu gguacugcau gcacgcaaug cuagcugccc 660cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua ugcucccacc 720uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca cgcagcaaug 780cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau uaaccuuuag 840caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu ucgugccagc 900cacacccucg agcuagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca uaugacuaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaa 102763187PRTArtificial sequenceModB FLT3L (human FLT3L in combination with a mouse optimized secretion sequence) 63Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser Thr Gln Asp Cys 20 25 30Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg 35 40 45Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser 50 55 60Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu65 70 75 80Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met 85 90 95Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys 100 105 110Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn 115 120 125Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys 130 135 140Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys145 150 155 160Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu 165 170 175Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Pro 180 185641120DNAArtificial sequenceModB FLT3L (5'UTR-CDS-3'UTR) 64ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatcca cccaggactg 240cagcttccag cactccccta tctcctccga cttcgccgtg aagatccggg agctgtccga 300ttacctgctg caggactacc ctgtgaccgt ggccagcaac ctgcaggacg aagaactgtg 360tggcggcctg tggcggctgg tgctggccca gcggtggatg gaacggctga aaaccgtggc 420cggctccaag atgcagggcc tgctcgagcg ggtgaacacc gagatccact tcgtgaccaa 480gtgcgccttc cagcctcctc cttcctgcct gcggttcgtg cagaccaaca tctcccggct 540gctgcaggaa acctccgagc agctggtcgc cctgaagcct tggatcaccc ggcagaactt 600ctcccggtgt ctggaactcc agtgtcagcc cgactcctcc accctgcctc ctccctggtc 660ccccaggcct ctggaagcca ccgcccctac cgccccacag cctccttgat aggtcgacgt 720cctggtactg catgcacgca atgctagctg cccctttccc gtcctgggta ccccgagtct 780cccccgacct cgggtcccag gtatgctccc acctccacct gccccactca ccacctctgc 840tagttccaga cacctcccaa gcacgcagca atgcagctca aaacgcttag cctagccaca 900cccccacggg aaacagcagt gattaacctt tagcaataaa cgaaagttta actaagctat 960actaacccca gggttggtca atttcgtgcc agccacaccc tcgagctagc aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa gcatatgact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1120651120RNAArtificial sequenceModB murine FLT3L 65ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggaucca cccaggacug 240cagcuuccag cacuccccua ucuccuccga cuucgccgug aagauccggg agcuguccga 300uuaccugcug caggacuacc cugugaccgu ggccagcaac cugcaggacg aagaacugug 360uggcggccug uggcggcugg ugcuggccca gcgguggaug gaacggcuga aaaccguggc 420cggcuccaag augcagggcc ugcucgagcg ggugaacacc gagauccacu ucgugaccaa 480gugcgccuuc cagccuccuc cuuccugccu gcgguucgug cagaccaaca ucucccggcu 540gcugcaggaa accuccgagc agcuggucgc ccugaagccu uggaucaccc ggcagaacuu 600cucccggugu cuggaacucc agugucagcc cgacuccucc acccugccuc cucccugguc 660ccccaggccu cuggaagcca ccgccccuac cgccccacag ccuccuugau aggucgacgu 720ccugguacug caugcacgca augcuagcug ccccuuuccc guccugggua ccccgagucu 780cccccgaccu cgggucccag guaugcuccc accuccaccu gccccacuca ccaccucugc 840uaguuccaga caccucccaa gcacgcagca augcagcuca aaacgcuuag ccuagccaca 900cccccacggg aaacagcagu gauuaaccuu uagcaauaaa cgaaaguuua acuaagcuau 960acuaacccca ggguugguca auuucgugcc agccacaccc ucgagcuagc aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa gcauaugacu aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 112066309PRTArtificial sequenceModA murine 41BBL 66Met Asp Gln His Thr Leu Asp Val Glu Asp Thr Ala Asp Ala Arg His1 5 10 15Pro Ala Gly Thr Ser Cys Pro Ser Asp Ala Ala Leu Leu Arg Asp Thr 20 25 30Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr 35 40 45Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg 50 55 60Asp Arg Glu Ala Ala Trp Pro Pro Ala Leu Asn Phe Cys Ser Arg His65 70 75 80Pro Lys Leu Tyr Gly Leu Val Ala Leu Val Leu Leu Leu Leu Ile Ala 85 90 95Ala Cys Val Pro Ile Phe Thr Arg Thr Glu Pro Arg Pro Ala Leu Thr 100 105 110Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln 115 120 125Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly 130 135 140Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys145 150 155 160Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr 165 170 175Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val 180 185 190Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu

Ser Pro 195 200 205Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val 210 215 220Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr225 230 235 240Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser 245 250 255Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly 260 265 270Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg Asp Trp Glu 275 280 285Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu Val Lys Pro 290 295 300Asp Asn Pro Trp Glu305671393DNAArtificial sequenceModA murine 41BBL (5'UTR-CDS-3'UTR) 67gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatggacca 60gcacacactt gatgtggagg ataccgcgga tgccagacat ccagcaggta cttcgtgccc 120ctcggatgcg gcgctcctca gagataccgg gctcctcgcg gacgctgcgc tcctctcaga 180tactgtgcgc cccacaaatg ccgcgctccc cacggatgct gcctaccctg cggttaatgt 240tcgggatcgc gaggccgcgt ggccgcctgc actgaacttc tgttcccgcc acccaaagct 300ctatggccta gtcgctttgg ttttgctgct tctgatcgcc gcctgtgttc ctatcttcac 360ccgcaccgag cctcggccag cgctcacaat caccacctcg cccaacctgg gtacccgaga 420gaataatgca gaccaggtca cccctgtttc ccacattggc tgccccaaca ctacacaaca 480gggctctcct gtgttcgcca agctactggc taaaaaccaa gcatcgttgt gcaatacaac 540tctgaactgg cacagccaag atggagctgg gagctcatac ctatctcaag gtctgaggta 600cgaagaagac aaaaaggagt tggtggtaga cagtcccggg ctctactacg tatttttgga 660actgaagctc agtccaacat tcacaaacac aggccacaag gtgcagggct gggtctctct 720tgttttgcaa gcaaagcctc aggtagatga ctttgacaac ttggccctga cagtggaact 780gttcccttgc tccatggaga acaagttagt ggaccgttcc tggagtcaac tgttgctcct 840gaaggctggc caccgcctca gtgtgggtct gagggcttat ctgcatggag cccaggatgc 900atacagagac tgggagctgt cttatcccaa caccaccagc tttggactct ttcttgtgaa 960acccgacaac ccatgggaat gatagggatc cgatctggta ctgcatgcac gcaatgctag 1020ctgccccttt cccgtcctgg gtaccccgag tctcccccga cctcgggtcc caggtatgct 1080cccacctcca cctgccccac tcaccacctc tgctagttcc agacacctcc caagcacgca 1140gcaatgcagc tcaaaacgct tagcctagcc acacccccac gggaaacagc agtgattaac 1200ctttagcaat aaacgaaagt ttaactaagc tatactaacc ccagggttgg tcaatttcgt 1260gccagccaca ccctcgagct agcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcatatg 1320actaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa aaa 1393681393RNAArtificial sequenceModA murine 41BBL 68gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauggacca 60gcacacacuu gauguggagg auaccgcgga ugccagacau ccagcaggua cuucgugccc 120cucggaugcg gcgcuccuca gagauaccgg gcuccucgcg gacgcugcgc uccucucaga 180uacugugcgc cccacaaaug ccgcgcuccc cacggaugcu gccuacccug cgguuaaugu 240ucgggaucgc gaggccgcgu ggccgccugc acugaacuuc uguucccgcc acccaaagcu 300cuauggccua gucgcuuugg uuuugcugcu ucugaucgcc gccuguguuc cuaucuucac 360ccgcaccgag ccucggccag cgcucacaau caccaccucg cccaaccugg guacccgaga 420gaauaaugca gaccagguca ccccuguuuc ccacauuggc ugccccaaca cuacacaaca 480gggcucuccu guguucgcca agcuacuggc uaaaaaccaa gcaucguugu gcaauacaac 540ucugaacugg cacagccaag auggagcugg gagcucauac cuaucucaag gucugaggua 600cgaagaagac aaaaaggagu uggugguaga cagucccggg cucuacuacg uauuuuugga 660acugaagcuc aguccaacau ucacaaacac aggccacaag gugcagggcu gggucucucu 720uguuuugcaa gcaaagccuc agguagauga cuuugacaac uuggcccuga caguggaacu 780guucccuugc uccauggaga acaaguuagu ggaccguucc uggagucaac uguugcuccu 840gaaggcuggc caccgccuca gugugggucu gagggcuuau cugcauggag cccaggaugc 900auacagagac ugggagcugu cuuaucccaa caccaccagc uuuggacucu uucuugugaa 960acccgacaac ccaugggaau gauagggauc cgaucuggua cugcaugcac gcaaugcuag 1020cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc cagguaugcu 1080cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc caagcacgca 1140gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc agugauuaac 1200cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg ucaauuucgu 1260gccagccaca cccucgagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcauaug 1320acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa aaa 139369309PRTArtificial sequenceModB murine 41BBL 69Met Asp Gln His Thr Leu Asp Val Glu Asp Thr Ala Asp Ala Arg His1 5 10 15Pro Ala Gly Thr Ser Cys Pro Ser Asp Ala Ala Leu Leu Arg Asp Thr 20 25 30Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr 35 40 45Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg 50 55 60Asp Arg Glu Ala Ala Trp Pro Pro Ala Leu Asn Phe Cys Ser Arg His65 70 75 80Pro Lys Leu Tyr Gly Leu Val Ala Leu Val Leu Leu Leu Leu Ile Ala 85 90 95Ala Cys Val Pro Ile Phe Thr Arg Thr Glu Pro Arg Pro Ala Leu Thr 100 105 110Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln 115 120 125Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly 130 135 140Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys145 150 155 160Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr 165 170 175Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val 180 185 190Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu Ser Pro 195 200 205Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val 210 215 220Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr225 230 235 240Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser 245 250 255Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly 260 265 270Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg Asp Trp Glu 275 280 285Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu Val Lys Pro 290 295 300Asp Asn Pro Trp Glu305701486DNAArtificial sequenceModB murine 41BBL (5'UTR-CDS-3'UTR) 70ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatgga ccagcacaca cttgatgtgg aggataccgc 180ggatgccaga catccagcag gtacttcgtg cccctcggat gcggcgctcc tcagagatac 240cgggctcctc gcggacgctg cgctcctctc agatactgtg cgccccacaa atgccgcgct 300ccccacggat gctgcctacc ctgcggttaa tgttcgggat cgcgaggccg cgtggccgcc 360tgcactgaac ttctgttccc gccacccaaa gctctatggc ctagtcgctt tggttttgct 420gcttctgatc gccgcctgtg ttcctatctt cacccgcacc gagcctcggc cagcgctcac 480aatcaccacc tcgcccaacc tgggtacccg agagaataat gcagaccagg tcacccctgt 540ttcccacatt ggctgcccca acactacaca acagggctct cctgtgttcg ccaagctact 600ggctaaaaac caagcatcgt tgtgcaatac aactctgaac tggcacagcc aagatggagc 660tgggagctca tacctatctc aaggtctgag gtacgaagaa gacaaaaagg agttggtggt 720agacagtccc gggctctact acgtattttt ggaactgaag ctcagtccaa cattcacaaa 780cacaggccac aaggtgcagg gctgggtctc tcttgttttg caagcaaagc ctcaggtaga 840tgactttgac aacttggccc tgacagtgga actgttccct tgctccatgg agaacaagtt 900agtggaccgt tcctggagtc aactgttgct cctgaaggct ggccaccgcc tcagtgtggg 960tctgagggct tatctgcatg gagcccagga tgcatacaga gactgggagc tgtcttatcc 1020caacaccacc agctttggac tctttcttgt gaaacccgac aacccatggg aatgataggg 1080atccgatctg gtactgcatg cacgcaatgc tagctgcccc tttcccgtcc tgggtacccc 1140gagtctcccc cgacctcggg tcccaggtat gctcccacct ccacctgccc cactcaccac 1200ctctgctagt tccagacacc tcccaagcac gcagcaatgc agctcaaaac gcttagccta 1260gccacacccc cacgggaaac agcagtgatt aacctttagc aataaacgaa agtttaacta 1320agctatacta accccagggt tggtcaattt cgtgccagcc acaccctcga gctagcaaaa 1380aaaaaaaaaa aaaaaaaaaa aaaaaagcat atgactaaaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1486711486RNAArtificial sequenceModB murine 41BBL 71ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccaugga ccagcacaca cuugaugugg aggauaccgc 180ggaugccaga cauccagcag guacuucgug ccccucggau gcggcgcucc ucagagauac 240cgggcuccuc gcggacgcug cgcuccucuc agauacugug cgccccacaa augccgcgcu 300ccccacggau gcugccuacc cugcgguuaa uguucgggau cgcgaggccg cguggccgcc 360ugcacugaac uucuguuccc gccacccaaa gcucuauggc cuagucgcuu ugguuuugcu 420gcuucugauc gccgccugug uuccuaucuu cacccgcacc gagccucggc cagcgcucac 480aaucaccacc ucgcccaacc uggguacccg agagaauaau gcagaccagg ucaccccugu 540uucccacauu ggcugcccca acacuacaca acagggcucu ccuguguucg ccaagcuacu 600ggcuaaaaac caagcaucgu ugugcaauac aacucugaac uggcacagcc aagauggagc 660ugggagcuca uaccuaucuc aaggucugag guacgaagaa gacaaaaagg aguugguggu 720agacaguccc gggcucuacu acguauuuuu ggaacugaag cucaguccaa cauucacaaa 780cacaggccac aaggugcagg gcugggucuc ucuuguuuug caagcaaagc cucagguaga 840ugacuuugac aacuuggccc ugacagugga acuguucccu ugcuccaugg agaacaaguu 900aguggaccgu uccuggaguc aacuguugcu ccugaaggcu ggccaccgcc ucaguguggg 960ucugagggcu uaucugcaug gagcccagga ugcauacaga gacugggagc ugucuuaucc 1020caacaccacc agcuuuggac ucuuucuugu gaaacccgac aacccauggg aaugauaggg 1080auccgaucug guacugcaug cacgcaaugc uagcugcccc uuucccgucc uggguacccc 1140gagucucccc cgaccucggg ucccagguau gcucccaccu ccaccugccc cacucaccac 1200cucugcuagu uccagacacc ucccaagcac gcagcaaugc agcucaaaac gcuuagccua 1260gccacacccc cacgggaaac agcagugauu aaccuuuagc aauaaacgaa aguuuaacua 1320agcuauacua accccagggu uggucaauuu cgugccagcc acacccucga gcuagcaaaa 1380aaaaaaaaaa aaaaaaaaaa aaaaaagcau augacuaaaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 148672935PRTArtificial sequenceModA murine CD27L-CD40L 72Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala1 5 10 15Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser His Pro Glu Pro 20 25 30His Thr Ala Glu Leu Gln Leu Asn Leu Thr Val Pro Arg Lys Asp Pro 35 40 45Thr Leu Arg Trp Gly Ala Gly Pro Ala Leu Gly Arg Ser Phe Thr His 50 55 60Gly Pro Glu Leu Glu Glu Gly His Leu Arg Ile His Gln Asp Gly Leu65 70 75 80Tyr Arg Leu His Ile Gln Val Thr Leu Ala Asn Cys Ser Ser Pro Gly 85 90 95Ser Thr Leu Gln His Arg Ala Thr Leu Ala Val Gly Ile Cys Ser Pro 100 105 110Ala Ala His Gly Ile Ser Leu Leu Arg Gly Arg Phe Gly Gln Asp Cys 115 120 125Thr Val Ala Leu Gln Arg Leu Thr Tyr Leu Val His Gly Asp Val Leu 130 135 140Cys Thr Asn Leu Thr Leu Pro Leu Leu Pro Ser Arg Asn Ala Asp Glu145 150 155 160Thr Phe Phe Gly Val Gln Trp Ile Cys Pro Gly Gly Gly Ser Gly Gly 165 170 175Gly His Pro Glu Pro His Thr Ala Glu Leu Gln Leu Asn Leu Thr Val 180 185 190Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala Gly Pro Ala Leu Gly 195 200 205Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu Gly His Leu Arg Ile 210 215 220His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln Val Thr Leu Ala Asn225 230 235 240Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg Ala Thr Leu Ala Val 245 250 255Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser Leu Leu Arg Gly Arg 260 265 270Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg Leu Thr Tyr Leu Val 275 280 285His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu Pro Leu Leu Pro Ser 290 295 300Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln Trp Ile Cys Pro Gly305 310 315 320Gly Gly Ser Gly Gly Gly His Pro Glu Pro His Thr Ala Glu Leu Gln 325 330 335Leu Asn Leu Thr Val Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala 340 345 350Gly Pro Ala Leu Gly Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu 355 360 365Gly His Leu Arg Ile His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln 370 375 380Val Thr Leu Ala Asn Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg385 390 395 400Ala Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser 405 410 415Leu Leu Arg Gly Arg Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg 420 425 430Leu Thr Tyr Leu Val His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu 435 440 445Pro Leu Leu Pro Ser Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln 450 455 460Trp Ile Cys Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly465 470 475 480Gly Gly Ser Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser 485 490 495Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly 500 505 510Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln 515 520 525Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr 530 535 540Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly545 550 555 560Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala 565 570 575Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His 580 585 590Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn 595 600 605Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe 610 615 620Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Gly Asp Glu Asp625 630 635 640Pro Gln Ile Ala Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala 645 650 655Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn 660 665 670Leu Val Met Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly 675 680 685Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro 690 695 700Ser Ser Gln Arg Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser705 710 715 720Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser 725 730 735Gln Leu Cys Glu Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu 740 745 750Gln Ala Gly Ala Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val 755 760 765Ile His Arg Val Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly 770 775 780Gly Ser Gly Gly Gly Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val785 790 795 800Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys 805 810 815Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly 820 825 830Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln 835 840 845Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile 850 855 860Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu865 870 875 880Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser 885 890 895Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe 900 905 910Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser 915 920 925Ser Phe Gly Leu Leu Lys Leu 930 935733281DNAArtificial sequenceModA murine CD27L-CD40L (5'UTR-CDS-3'UTR) 73gggcgaacta gtattcttct ggtccccaca gactcagaga gaacccgcca ccatgagagt 60gaccgccccc agaaccctga tcctgctgct gtctggcgcc ctggccctga cagagacatg 120ggccggaagc ggatcccacc ccgagcccca caccgccgaa ctgcagctga acctgaccgt 180gcccagaaag gaccccaccc tgagatgggg agctggccct gctctgggca gatcctttac 240acacggcccc gagctggaag aaggccacct gagaatccac caggacggcc tgtacagact 300gcacatccaa gtgaccctgg ccaactgcag cagccctggc tctaccctgc agcacagagc 360cacactggcc gtgggcatct gtagccctgc tgctcacgga atcagcctgc tgagaggcag 420attcggccag gactgtaccg tggccctgca gaggctgacc tatctggtgc atggcgacgt 480gctgtgcacc aacctgacac tgcctctgct gcccagcaga aacgccgacg aaacattctt 540tggagtgcag tggatttgtc ctggcggagg gtccggggga ggacacccag aacctcatac 600agctgaactg cagctgaacc tgaccgtgcc

cagaaaggac cccaccctga gatggggagc 660tggccctgct ctgggcagat cctttacaca cggccccgag ctggaagaag gccacctgag 720aatccaccag gacggcctgt acagactgca catccaagtg accctggcca actgcagcag 780ccctggctct accctgcagc acagagccac actggccgtg ggcatctgta gccctgctgc 840tcacggaatc agcctgctga gaggcagatt cggccaggac tgtaccgtgg ccctgcagag 900gctgacctat ctggtgcatg gcgacgtgct gtgcaccaac ctgacactgc ctctgctgcc 960cagcagaaac gccgacgaaa cattctttgg agtgcagtgg atttgtcctg ggggaggctc 1020cggaggcgga caccctgaac ctcatacagc tgaactgcag ctgaacctga ccgtgcccag 1080aaaggacccc accctgagat ggggagctgg ccctgctctg ggcagatcct ttacacacgg 1140ccccgagctg gaagaaggcc acctgagaat ccaccaggac ggcctgtaca gactgcacat 1200ccaagtgacc ctggccaact gcagcagccc tggctctacc ctgcagcaca gagccacact 1260ggccgtgggc atctgtagcc ctgctgctca cggaatcagc ctgctgagag gcagattcgg 1320ccaggactgt accgtggccc tgcagaggct gacctatctg gtgcatggcg acgtgctgtg 1380caccaacctg acactgcctc tgctgcccag cagaaacgcc gacgagacct tcttcggcgt 1440ccagtggatc tgccccggag gcggtggtag tggaggtggc gggtccggtg gaggtggaag 1500cggcgacgag gacccccaga tcgccgccca cgtggtgtct gaggccaaca gcaacgccgc 1560ctctgtgctg cagtgggcca agaaaggcta ctacaccatg aagtccaacc tcgtgatgct 1620ggaaaacggc aagcagctga ccgtgaagcg cgagggcctg tactatgtgt acacccaagt 1680gacattctgc agcaaccgcg agcccagcag ccagaggcct tttatcgtgg gcctgtggct 1740gaagcctagc agcggcagcg agagaatcct gctgaaggcc gccaacaccc acagcagctc 1800tcagctgtgc gagcagcagt ctgtgcacct gggaggcgtg ttcgagctgc aagctggcgc 1860ttccgtgttc gtgaacgtga ccgaggccag ccaagtgatc cacagagtgg gcttcagcag 1920ctttggactg ctcaaactgg gcggagggtc cggcggaggc ggagatgaag atcctcagat 1980tgctgcccac gtggtgtctg aggccaacag caacgccgcc tctgtgctgc agtgggccaa 2040gaaaggctac tacaccatga agtccaacct cgtgatgctg gaaaacggca agcagctgac 2100cgtgaagcgc gagggcctgt actatgtgta cacccaagtg acattctgca gcaaccgcga 2160gcccagcagc cagaggcctt ttatcgtggg cctgtggctg aagcctagca gcggcagcga 2220gagaatcctg ctgaaggccg ccaacaccca cagcagctct cagctgtgcg agcagcagtc 2280tgtgcacctg ggaggcgtgt tcgagctgca agctggcgct tccgtgttcg tgaacgtgac 2340cgaggccagc caagtgatcc acagagtggg cttcagcagc tttggactgc tcaaactggg 2400aggcggctcc ggaggcggag gagatgaaga tcctcagatt gctgcccacg tggtgtctga 2460ggccaacagc aacgccgcct ctgtgctgca gtgggccaag aaaggctact acaccatgaa 2520gtccaacctc gtgatgctgg aaaacggcaa gcagctgacc gtgaagcgcg agggcctgta 2580ctatgtgtac acccaagtga cattctgcag caaccgcgag cccagcagcc agaggccttt 2640tatcgtgggc ctgtggctga agcctagcag cggcagcgag agaatcctgc tgaaggccgc 2700caacacccac agcagctctc agctgtgcga gcagcagtct gtgcacctgg gaggcgtgtt 2760cgagctgcaa gctggcgctt ccgtgttcgt gaacgtgacc gaggccagcc aagtgatcca 2820cagagtgggc ttctcctcct tcggcctcct gaagctgtga ctcgagagct cgctttcttg 2880ctgtccaatt tctattaaag gttcctttgt tccctaagtc caactactaa actgggggat 2940attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat tttcattgct 3000gcgtcgagag ctcgctttct tgctgtccaa tttctattaa aggttccttt gttccctaag 3060tccaactact aaactggggg atattatgaa gggccttgag catctggatt ctgcctaata 3120aaaaacattt attttcattg ctgcgtcgag acctggtcca gagtcgctag caaaaaaaaa 3180aaaaaaaaaa aaaaaaaaaa agcatatgac taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 3281743281RNAArtificial sequenceModA murine CD27L-CD40L 74gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60gaccgccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120ggccggaagc ggaucccacc ccgagcccca caccgccgaa cugcagcuga accugaccgu 180gcccagaaag gaccccaccc ugagaugggg agcuggcccu gcucugggca gauccuuuac 240acacggcccc gagcuggaag aaggccaccu gagaauccac caggacggcc uguacagacu 300gcacauccaa gugacccugg ccaacugcag cagcccuggc ucuacccugc agcacagagc 360cacacuggcc gugggcaucu guagcccugc ugcucacgga aucagccugc ugagaggcag 420auucggccag gacuguaccg uggcccugca gaggcugacc uaucuggugc auggcgacgu 480gcugugcacc aaccugacac ugccucugcu gcccagcaga aacgccgacg aaacauucuu 540uggagugcag uggauuuguc cuggcggagg guccggggga ggacacccag aaccucauac 600agcugaacug cagcugaacc ugaccgugcc cagaaaggac cccacccuga gauggggagc 660uggcccugcu cugggcagau ccuuuacaca cggccccgag cuggaagaag gccaccugag 720aauccaccag gacggccugu acagacugca cauccaagug acccuggcca acugcagcag 780cccuggcucu acccugcagc acagagccac acuggccgug ggcaucugua gcccugcugc 840ucacggaauc agccugcuga gaggcagauu cggccaggac uguaccgugg cccugcagag 900gcugaccuau cuggugcaug gcgacgugcu gugcaccaac cugacacugc cucugcugcc 960cagcagaaac gccgacgaaa cauucuuugg agugcagugg auuuguccug ggggaggcuc 1020cggaggcgga cacccugaac cucauacagc ugaacugcag cugaaccuga ccgugcccag 1080aaaggacccc acccugagau ggggagcugg cccugcucug ggcagauccu uuacacacgg 1140ccccgagcug gaagaaggcc accugagaau ccaccaggac ggccuguaca gacugcacau 1200ccaagugacc cuggccaacu gcagcagccc uggcucuacc cugcagcaca gagccacacu 1260ggccgugggc aucuguagcc cugcugcuca cggaaucagc cugcugagag gcagauucgg 1320ccaggacugu accguggccc ugcagaggcu gaccuaucug gugcauggcg acgugcugug 1380caccaaccug acacugccuc ugcugcccag cagaaacgcc gacgagaccu ucuucggcgu 1440ccaguggauc ugccccggag gcggugguag uggagguggc ggguccggug gagguggaag 1500cggcgacgag gacccccaga ucgccgccca cguggugucu gaggccaaca gcaacgccgc 1560cucugugcug cagugggcca agaaaggcua cuacaccaug aaguccaacc ucgugaugcu 1620ggaaaacggc aagcagcuga ccgugaagcg cgagggccug uacuaugugu acacccaagu 1680gacauucugc agcaaccgcg agcccagcag ccagaggccu uuuaucgugg gccuguggcu 1740gaagccuagc agcggcagcg agagaauccu gcugaaggcc gccaacaccc acagcagcuc 1800ucagcugugc gagcagcagu cugugcaccu gggaggcgug uucgagcugc aagcuggcgc 1860uuccguguuc gugaacguga ccgaggccag ccaagugauc cacagagugg gcuucagcag 1920cuuuggacug cucaaacugg gcggaggguc cggcggaggc ggagaugaag auccucagau 1980ugcugcccac guggugucug aggccaacag caacgccgcc ucugugcugc agugggccaa 2040gaaaggcuac uacaccauga aguccaaccu cgugaugcug gaaaacggca agcagcugac 2100cgugaagcgc gagggccugu acuaugugua cacccaagug acauucugca gcaaccgcga 2160gcccagcagc cagaggccuu uuaucguggg ccuguggcug aagccuagca gcggcagcga 2220gagaauccug cugaaggccg ccaacaccca cagcagcucu cagcugugcg agcagcaguc 2280ugugcaccug ggaggcgugu ucgagcugca agcuggcgcu uccguguucg ugaacgugac 2340cgaggccagc caagugaucc acagaguggg cuucagcagc uuuggacugc ucaaacuggg 2400aggcggcucc ggaggcggag gagaugaaga uccucagauu gcugcccacg uggugucuga 2460ggccaacagc aacgccgccu cugugcugca gugggccaag aaaggcuacu acaccaugaa 2520guccaaccuc gugaugcugg aaaacggcaa gcagcugacc gugaagcgcg agggccugua 2580cuauguguac acccaaguga cauucugcag caaccgcgag cccagcagcc agaggccuuu 2640uaucgugggc cuguggcuga agccuagcag cggcagcgag agaauccugc ugaaggccgc 2700caacacccac agcagcucuc agcugugcga gcagcagucu gugcaccugg gaggcguguu 2760cgagcugcaa gcuggcgcuu ccguguucgu gaacgugacc gaggccagcc aagugaucca 2820cagagugggc uucuccuccu ucggccuccu gaagcuguga cucgagagcu cgcuuucuug 2880cuguccaauu ucuauuaaag guuccuuugu ucccuaaguc caacuacuaa acugggggau 2940auuaugaagg gccuugagca ucuggauucu gccuaauaaa aaacauuuau uuucauugcu 3000gcgucgagag cucgcuuucu ugcuguccaa uuucuauuaa agguuccuuu guucccuaag 3060uccaacuacu aaacuggggg auauuaugaa gggccuugag caucuggauu cugccuaaua 3120aaaaacauuu auuuucauug cugcgucgag accuggucca gagucgcuag caaaaaaaaa 3180aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 328175935PRTArtificial sequenceModB murine CD27L-CD40L 75Met Gly Ala Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ala Ala Ala1 5 10 15Leu Ala Pro Thr Gln Thr Arg Ala Gly Pro Gly Ser His Pro Glu Pro 20 25 30His Thr Ala Glu Leu Gln Leu Asn Leu Thr Val Pro Arg Lys Asp Pro 35 40 45Thr Leu Arg Trp Gly Ala Gly Pro Ala Leu Gly Arg Ser Phe Thr His 50 55 60Gly Pro Glu Leu Glu Glu Gly His Leu Arg Ile His Gln Asp Gly Leu65 70 75 80Tyr Arg Leu His Ile Gln Val Thr Leu Ala Asn Cys Ser Ser Pro Gly 85 90 95Ser Thr Leu Gln His Arg Ala Thr Leu Ala Val Gly Ile Cys Ser Pro 100 105 110Ala Ala His Gly Ile Ser Leu Leu Arg Gly Arg Phe Gly Gln Asp Cys 115 120 125Thr Val Ala Leu Gln Arg Leu Thr Tyr Leu Val His Gly Asp Val Leu 130 135 140Cys Thr Asn Leu Thr Leu Pro Leu Leu Pro Ser Arg Asn Ala Asp Glu145 150 155 160Thr Phe Phe Gly Val Gln Trp Ile Cys Pro Gly Gly Gly Ser Gly Gly 165 170 175Gly His Pro Glu Pro His Thr Ala Glu Leu Gln Leu Asn Leu Thr Val 180 185 190Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala Gly Pro Ala Leu Gly 195 200 205Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu Gly His Leu Arg Ile 210 215 220His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln Val Thr Leu Ala Asn225 230 235 240Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg Ala Thr Leu Ala Val 245 250 255Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser Leu Leu Arg Gly Arg 260 265 270Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg Leu Thr Tyr Leu Val 275 280 285His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu Pro Leu Leu Pro Ser 290 295 300Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln Trp Ile Cys Pro Gly305 310 315 320Gly Gly Ser Gly Gly Gly His Pro Glu Pro His Thr Ala Glu Leu Gln 325 330 335Leu Asn Leu Thr Val Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala 340 345 350Gly Pro Ala Leu Gly Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu 355 360 365Gly His Leu Arg Ile His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln 370 375 380Val Thr Leu Ala Asn Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg385 390 395 400Ala Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser 405 410 415Leu Leu Arg Gly Arg Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg 420 425 430Leu Thr Tyr Leu Val His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu 435 440 445Pro Leu Leu Pro Ser Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln 450 455 460Trp Ile Cys Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly465 470 475 480Gly Gly Ser Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser 485 490 495Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly 500 505 510Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln 515 520 525Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr 530 535 540Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly545 550 555 560Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala 565 570 575Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His 580 585 590Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn 595 600 605Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe 610 615 620Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Gly Asp Glu Asp625 630 635 640Pro Gln Ile Ala Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala 645 650 655Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn 660 665 670Leu Val Met Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly 675 680 685Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro 690 695 700Ser Ser Gln Arg Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser705 710 715 720Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser 725 730 735Gln Leu Cys Glu Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu 740 745 750Gln Ala Gly Ala Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val 755 760 765Ile His Arg Val Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly 770 775 780Gly Ser Gly Gly Gly Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val785 790 795 800Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys 805 810 815Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly 820 825 830Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln 835 840 845Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile 850 855 860Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu865 870 875 880Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser 885 890 895Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe 900 905 910Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser 915 920 925Ser Phe Gly Leu Leu Lys Leu 930 935763361DNAArtificial sequenceModB murine CD27L-CD40L (5'UTR-CDS-3'UTR) 76ggaataaact agtctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat 60tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa 120ttttcaccat ttacgaacga tagccatggg cgccatggcc cctagaacat tgctcctgct 180gctggccgct gccctggccc ctacacagac aagagctgga cctggatccc accccgagcc 240ccacaccgcc gaactgcagc tgaacctgac cgtgcccaga aaggacccca ccctgagatg 300gggagctggc cctgctctgg gcagatcctt tacacacggc cccgagctgg aagaaggcca 360cctgagaatc caccaggacg gcctgtacag actgcacatc caagtgaccc tggccaactg 420cagcagccct ggctctaccc tgcagcacag agccacactg gccgtgggca tctgtagccc 480tgctgctcac ggaatcagcc tgctgagagg cagattcggc caggactgta ccgtggccct 540gcagaggctg acctatctgg tgcatggcga cgtgctgtgc accaacctga cactgcctct 600gctgcccagc agaaacgccg acgaaacatt ctttggagtg cagtggattt gtcctggcgg 660agggtccggg ggaggacacc cagaacctca tacagctgaa ctgcagctga acctgaccgt 720gcccagaaag gaccccaccc tgagatgggg agctggccct gctctgggca gatcctttac 780acacggcccc gagctggaag aaggccacct gagaatccac caggacggcc tgtacagact 840gcacatccaa gtgaccctgg ccaactgcag cagccctggc tctaccctgc agcacagagc 900cacactggcc gtgggcatct gtagccctgc tgctcacgga atcagcctgc tgagaggcag 960attcggccag gactgtaccg tggccctgca gaggctgacc tatctggtgc atggcgacgt 1020gctgtgcacc aacctgacac tgcctctgct gcccagcaga aacgccgacg aaacattctt 1080tggagtgcag tggatttgtc ctgggggagg ctccggaggc ggacaccctg aacctcatac 1140agctgaactg cagctgaacc tgaccgtgcc cagaaaggac cccaccctga gatggggagc 1200tggccctgct ctgggcagat cctttacaca cggccccgag ctggaagaag gccacctgag 1260aatccaccag gacggcctgt acagactgca catccaagtg accctggcca actgcagcag 1320ccctggctct accctgcagc acagagccac actggccgtg ggcatctgta gccctgctgc 1380tcacggaatc agcctgctga gaggcagatt cggccaggac tgtaccgtgg ccctgcagag 1440gctgacctat ctggtgcatg gcgacgtgct gtgcaccaac ctgacactgc ctctgctgcc 1500cagcagaaac gccgacgaga ccttcttcgg cgtccagtgg atctgccccg gaggcggtgg 1560tagtggaggt ggcgggtccg gtggaggtgg aagcggcgac gaggaccccc agatcgccgc 1620ccacgtggtg tctgaggcca acagcaacgc cgcctctgtg ctgcagtggg ccaagaaagg 1680ctactacacc atgaagtcca acctcgtgat gctggaaaac ggcaagcagc tgaccgtgaa 1740gcgcgagggc ctgtactatg tgtacaccca agtgacattc tgcagcaacc gcgagcccag 1800cagccagagg ccttttatcg tgggcctgtg gctgaagcct agcagcggca gcgagagaat 1860cctgctgaag gccgccaaca cccacagcag ctctcagctg tgcgagcagc agtctgtgca 1920cctgggaggc gtgttcgagc tgcaagctgg cgcttccgtg ttcgtgaacg tgaccgaggc 1980cagccaagtg atccacagag tgggcttcag cagctttgga ctgctcaaac tgggcggagg 2040gtccggcgga ggcggagatg aagatcctca gattgctgcc cacgtggtgt ctgaggccaa 2100cagcaacgcc gcctctgtgc tgcagtgggc caagaaaggc tactacacca tgaagtccaa 2160cctcgtgatg ctggaaaacg gcaagcagct gaccgtgaag cgcgagggcc tgtactatgt 2220gtacacccaa gtgacattct gcagcaaccg cgagcccagc agccagaggc cttttatcgt 2280gggcctgtgg ctgaagccta gcagcggcag cgagagaatc ctgctgaagg ccgccaacac 2340ccacagcagc tctcagctgt gcgagcagca gtctgtgcac ctgggaggcg tgttcgagct 2400gcaagctggc gcttccgtgt tcgtgaacgt gaccgaggcc agccaagtga tccacagagt 2460gggcttcagc agctttggac tgctcaaact gggaggcggc tccggaggcg gaggagatga 2520agatcctcag attgctgccc acgtggtgtc tgaggccaac agcaacgccg cctctgtgct 2580gcagtgggcc aagaaaggct actacaccat gaagtccaac ctcgtgatgc tggaaaacgg 2640caagcagctg accgtgaagc gcgagggcct gtactatgtg tacacccaag tgacattctg 2700cagcaaccgc gagcccagca gccagaggcc ttttatcgtg ggcctgtggc tgaagcctag 2760cagcggcagc gagagaatcc tgctgaaggc cgccaacacc cacagcagct ctcagctgtg 2820cgagcagcag tctgtgcacc tgggaggcgt gttcgagctg caagctggcg cttccgtgtt 2880cgtgaacgtg accgaggcca gccaagtgat ccacagagtg ggcttctcct ccttcggcct 2940cctgaagctg tgactcgacg tcctggtact gcatgcacgc aatgctagct gcccctttcc 3000cgtcctgggt accccgagtc tcccccgacc tcgggtccca ggtatgctcc cacctccacc 3060tgccccactc accacctctg ctagttccag acacctccca agcacgcagc aatgcagctc 3120aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 3180acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccacacc

3240ctcgagctag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcatatgac taaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360a 3361773361RNAArtificial sequenceModB murine CD27L-CD40L 77ggaauaaacu agucucaaca caacauauac aaaacaaacg aaucucaagc aaucaagcau 60ucuacuucua uugcagcaau uuaaaucauu ucuuuuaaag caaaagcaau uuucugaaaa 120uuuucaccau uuacgaacga uagccauggg cgccauggcc ccuagaacau ugcuccugcu 180gcuggccgcu gcccuggccc cuacacagac aagagcugga ccuggauccc accccgagcc 240ccacaccgcc gaacugcagc ugaaccugac cgugcccaga aaggacccca cccugagaug 300gggagcuggc ccugcucugg gcagauccuu uacacacggc cccgagcugg aagaaggcca 360ccugagaauc caccaggacg gccuguacag acugcacauc caagugaccc uggccaacug 420cagcagcccu ggcucuaccc ugcagcacag agccacacug gccgugggca ucuguagccc 480ugcugcucac ggaaucagcc ugcugagagg cagauucggc caggacugua ccguggcccu 540gcagaggcug accuaucugg ugcauggcga cgugcugugc accaaccuga cacugccucu 600gcugcccagc agaaacgccg acgaaacauu cuuuggagug caguggauuu guccuggcgg 660aggguccggg ggaggacacc cagaaccuca uacagcugaa cugcagcuga accugaccgu 720gcccagaaag gaccccaccc ugagaugggg agcuggcccu gcucugggca gauccuuuac 780acacggcccc gagcuggaag aaggccaccu gagaauccac caggacggcc uguacagacu 840gcacauccaa gugacccugg ccaacugcag cagcccuggc ucuacccugc agcacagagc 900cacacuggcc gugggcaucu guagcccugc ugcucacgga aucagccugc ugagaggcag 960auucggccag gacuguaccg uggcccugca gaggcugacc uaucuggugc auggcgacgu 1020gcugugcacc aaccugacac ugccucugcu gcccagcaga aacgccgacg aaacauucuu 1080uggagugcag uggauuuguc cugggggagg cuccggaggc ggacacccug aaccucauac 1140agcugaacug cagcugaacc ugaccgugcc cagaaaggac cccacccuga gauggggagc 1200uggcccugcu cugggcagau ccuuuacaca cggccccgag cuggaagaag gccaccugag 1260aauccaccag gacggccugu acagacugca cauccaagug acccuggcca acugcagcag 1320cccuggcucu acccugcagc acagagccac acuggccgug ggcaucugua gcccugcugc 1380ucacggaauc agccugcuga gaggcagauu cggccaggac uguaccgugg cccugcagag 1440gcugaccuau cuggugcaug gcgacgugcu gugcaccaac cugacacugc cucugcugcc 1500cagcagaaac gccgacgaga ccuucuucgg cguccagugg aucugccccg gaggcggugg 1560uaguggaggu ggcggguccg guggaggugg aagcggcgac gaggaccccc agaucgccgc 1620ccacguggug ucugaggcca acagcaacgc cgccucugug cugcaguggg ccaagaaagg 1680cuacuacacc augaagucca accucgugau gcuggaaaac ggcaagcagc ugaccgugaa 1740gcgcgagggc cuguacuaug uguacaccca agugacauuc ugcagcaacc gcgagcccag 1800cagccagagg ccuuuuaucg ugggccugug gcugaagccu agcagcggca gcgagagaau 1860ccugcugaag gccgccaaca cccacagcag cucucagcug ugcgagcagc agucugugca 1920ccugggaggc guguucgagc ugcaagcugg cgcuuccgug uucgugaacg ugaccgaggc 1980cagccaagug auccacagag ugggcuucag cagcuuugga cugcucaaac ugggcggagg 2040guccggcgga ggcggagaug aagauccuca gauugcugcc cacguggugu cugaggccaa 2100cagcaacgcc gccucugugc ugcagugggc caagaaaggc uacuacacca ugaaguccaa 2160ccucgugaug cuggaaaacg gcaagcagcu gaccgugaag cgcgagggcc uguacuaugu 2220guacacccaa gugacauucu gcagcaaccg cgagcccagc agccagaggc cuuuuaucgu 2280gggccugugg cugaagccua gcagcggcag cgagagaauc cugcugaagg ccgccaacac 2340ccacagcagc ucucagcugu gcgagcagca gucugugcac cugggaggcg uguucgagcu 2400gcaagcuggc gcuuccgugu ucgugaacgu gaccgaggcc agccaaguga uccacagagu 2460gggcuucagc agcuuuggac ugcucaaacu gggaggcggc uccggaggcg gaggagauga 2520agauccucag auugcugccc acgugguguc ugaggccaac agcaacgccg ccucugugcu 2580gcagugggcc aagaaaggcu acuacaccau gaaguccaac cucgugaugc uggaaaacgg 2640caagcagcug accgugaagc gcgagggccu guacuaugug uacacccaag ugacauucug 2700cagcaaccgc gagcccagca gccagaggcc uuuuaucgug ggccuguggc ugaagccuag 2760cagcggcagc gagagaaucc ugcugaaggc cgccaacacc cacagcagcu cucagcugug 2820cgagcagcag ucugugcacc ugggaggcgu guucgagcug caagcuggcg cuuccguguu 2880cgugaacgug accgaggcca gccaagugau ccacagagug ggcuucuccu ccuucggccu 2940ccugaagcug ugacucgacg uccugguacu gcaugcacgc aaugcuagcu gccccuuucc 3000cguccugggu accccgaguc ucccccgacc ucggguccca gguaugcucc caccuccacc 3060ugccccacuc accaccucug cuaguuccag acaccuccca agcacgcagc aaugcagcuc 3120aaaacgcuua gccuagccac acccccacgg gaaacagcag ugauuaaccu uuagcaauaa 3180acgaaaguuu aacuaagcua uacuaacccc aggguugguc aauuucgugc cagccacacc 3240cucgagcuag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360a 336178110RNAArtificial sequencePoly-A 78aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcauaugacu aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11079444PRTArtificial sequenceAnti-PD1 Mab heavy chain 79Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Gly Gly Gly Arg Asp Thr Tyr Phe Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 44080214PRTArtificial sequenceAnti-PD1 Mab light chain 80Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Ser Ile Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Asn Thr Phe 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu His Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Thr Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Asn Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Val Val Asp Phe Arg Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210818PRTArtificial sequenceHCDR1 81Gly Phe Thr Phe Ser Asn Phe Gly1 5828PRTArtificial sequenceHCDR2 82Ile Ser Gly Gly Gly Arg Asp Thr1 58310PRTArtificial sequenceHCDR3 83Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr1 5 10846PRTArtificial sequenceLCDR1 84Leu Ser Ile Asn Thr Phe1 5853PRTArtificial sequenceLCDR2 85Ala Ala Ser1869PRTArtificial sequenceLCDR3 86Gln Gln Ser Ser Asn Thr Pro Phe Thr1 587117PRTArtificial sequenceAnti-PD1 Mab VH 87Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Gly Gly Gly Arg Asp Thr Tyr Phe Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser 11588107PRTArtificial sequenceAnti-PD1 Mab VL 88Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Ser Ile Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Asn Thr Phe 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu His Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Thr Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Asn Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Val Val Asp Phe Arg 100 105

Claims

1.-88. (canceled)

89. A composition comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein.

90. The composition of claim 89, wherein the IFN.alpha. protein is an IFN.alpha.2b protein.

91. The composition of claim 89, 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 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 80% identity to the amino acid sequence of SEQ ID NO: 14.

92. The composition of claim 89, 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 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 80% identity to the amino acid sequence of SEQ ID NO: 24.

93. The composition of claim 89, wherein (i) the RNA encoding an IFN.alpha. protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFN.alpha. protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 19.

94. The composition of claim 89, 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 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 80% identity to the amino acid sequence of SEQ ID NO: 27.

95. The composition of claim 89, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine, wherein the modified nucleoside is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.), 5-methyl-uridine (m5U), or a combination thereof.

96. The composition of claim 89, wherein each RNA comprises a modified nucleoside in place of at least one uridine, wherein the modified nucleoside is pseudouridine (.psi.), N1-methyl-pseudouridine (m1.psi.), 5-methyl-uridine (m5U), or a combination thereof.

97. The composition of claim 89, wherein at least one RNA comprises the 5' cap m.sub.2.sup.7,3'-OGppp(m1.sup.2'-OApG, or 3'-O-Me-m.sup.7G(5')ppp(5')G, or m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG, or 3'-O-Me-m.sup.7G(5')ppp(5')G.

98. The composition of claim 89, wherein at least one RNA comprises 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 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6 and/or a 3' UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 8.

99. The composition of claim 89, wherein at least one RNA comprises a poly-A tail of at least 100 nucleotides.

100. The composition of claim 99, wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 78.

101. The composition of claim 89, wherein one or more RNA comprises: a 5' cap comprising)m.sub.2.sup.7,3'-OGppp(m.sub.1.sup.2'-O)ApG or 3'-O-Me-m.sup.7G(5')ppp(5')G; 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 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6; a 3' UTR comprising (i) the nucleotide sequence of SEQ ID NO: 8, or (ii) a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:8; and a poly-A tail comprising at least 100 nucleotides.

102. The composition of claim 101, wherein the poly-A tail comprises SEQ ID NO: 78.

103. A pharmaceutical composition comprising the composition of claim 89 and a pharmaceutically acceptable carrier, diluent and/or excipient.

104. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the composition of claim 89.

105. 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 IFN.alpha. protein, or a GM-CSF protein, and additional RNA, wherein: if the first RNA encodes an IL-12sc protein, then the additional RNA encodes an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein; if the first RNA encodes an IL-15 sushi protein, then the additional RNA encodes an IL-12sc protein, an IFN.alpha. protein, and a GM-CSF protein; if the first RNA encodes an IFN.alpha. protein, then the additional RNA encodes an IL-15 sushi protein, an IL-12sc protein, and a GM-CSF protein; and if the first RNA encodes a GM-CSF protein, then the additional RNA encodes an IL-15 sushi protein, an IFN.alpha. protein, and an IL-12sc protein.

106. The method of claim 105, wherein the first RNA is administered to the subject at the same time as the additional RNA.

107. The method of claim 104, wherein the RNA is administered intra-tumorally or peri-tumorally.

108. The method of claim 104, wherein the subject is further treated with an additional therapy comprising (i) surgery to excise, resect, or debulk a tumor, (ii) immunotherapy, (iii) radiotherapy, or (iv) chemotherapy.

109. The method of claim 104, wherein the subject is further treated with a checkpoint modulator.

110. The method of claim 109, 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.

111. The method of claim 109, wherein the RNA is administered intra-tumorally or peri-tumorally via injection, and the checkpoint modulator is administered systemically.

112. The method of claim 104, wherein the solid tumor is a sarcoma, carcinoma, or lymphoma.

113. The method of claim 104, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.

114. The method of claim 104, 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.

115. The method of claim 104, wherein treating or reducing the likelihood of the solid tumor comprises reducing the size of a tumor, reducing the likelihood of a reoccurrence of cancer in remission, or reducing the likelihood of cancer metastasis in the subject.

116. A method for treating or reducing the likelihood of a solid tumor, comprising administering to a subject in need thereof RNAs and a further therapy, wherein the RNAs encode an IL-12sc protein, an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein, and the further therapy comprises immunotherapy, chemotherapy, or a checkpoint modulator.

117. An isolated nucleic acid comprising a sequence encoding an IL-12sc protein, wherein: the sequence encoding the IL-12sc protein comprises a) contiguous nucleotides having at least 78% identity to nucleotides 1-984 of SEQ ID NO: 16 or 18, b) contiguous nucleotides having at least 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16 or 18, and c) nucleotides encoding a linker between the nucleotides of a) and b).

118. An isolated nucleic acid comprising a sequence encoding an IFN.alpha. protein, wherein: the sequence encoding the IFN.alpha. protein has at least 80% identity to the nucleotide sequence of SEQ ID NO: 21 or 23.

119. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the nucleic acid of claim 117, wherein the nucleic acid is RNA, and wherein: the subject is further treated with additional RNA encoding an IL-15 sushi protein, an IFN.alpha. protein, and a GM-CSF protein.

120. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof the nucleic of claim 118, wherein the nucleic acid is RNA, and wherein: the subject is further treated with additional RNA encoding an IL-15 sushi protein, an IL-12sc protein, and a GM-CSF protein.

121. A method of producing an RNA encoding IL-12sc, comprising contacting an expression construct comprising the DNA nucleic acid of claim 117 operably linked to a promoter with an RNA polymerase under conditions permissive for transcription.

122. A method of producing an RNA encoding IFN.alpha., comprising contacting an expression construct comprising the DNA nucleic acid of claim 118 operably linked to a promoter with an RNA polymerase under conditions permissive for transcription.

123. An expression construct comprising the nucleic acid of claim 117 operably linked to a promoter.

124. An expression construct comprising the nucleic acid of claim 118 operably linked to a promoter.

125. A kit comprising the composition of claim 89.

126. A kit comprising RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein, wherein the RNAs are not in the same container.

127. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein, wherein the solid tumor is in a lymph node.

128. A method for treating or reducing the likelihood of a solid tumor comprising administering to a subject in need thereof RNA encoding an IL-12sc protein, RNA encoding an IL-15 sushi protein, RNA encoding an IFN.alpha. protein, and RNA encoding a GM-CSF protein, wherein the RNA is administered via intra- or peri-tumoral injection into a first tumor, and wherein the injected first tumor and a non-injected second tumor are both reduced in size.

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