Title of Invention

FUSION PROTEIN MOLECULES AND METHOD OF USE

This present invention claims priority to U.S. Provisional Application No. 61/802,169, filed on March 15, 2013. This present invention also claims priority to U.S. Provisional Application No. 61/655,428, filed on June 4, 2012, now abandoned. Both of these applications are incorporated herein in their entirety by reference.

Technical Field

The present invention relates to fusion molecules comprising a cytokine moiety and a targeting moiety. The cytokine moiety comprises one or more cytokines. The targeting moiety comprises one or more ligands. Each ligand is a peptide, a protein, or a small molecule that recognizes a receptor on the target, such as but not limited to cells or viral particles.

Background Art

Cytokines, such as lymphokines, chemokines, and growth factors, are produced by various types of cells, including lymphocytes and monocytes, and play a wide range of roles in human physiology and disease. Several cytokines have been shown that when used clinically they are efficacious for treating various diseases.

For example, interleukin-2 (IL-2), a cytokine, has been approved by the FDA in US and other agencies throughout the world for clinical use to treat metastatic renal cell carcinoma and malignant metastatic melanoma. However, the currently marketed IL-2 has a limited therapeutic window due to side effects, such as capillary leak syndrome (CLS). IL-2 works by enhancing proliferation of T cells through out the circulation, not specifically at the site of tumors, including both primary and metastatic tumors. Therefore, therapeutic window can be increased if IL-2 can be targeted to tumor cells.

U.S. Patent USRE33653E describes composition of a mutant IL-2 and method of use. US4935356A describes IL-2 expression and purification in E. coli. US8580267B2 discloses immunocytokines for tumour therapy with chemotherapeutic agents, comprising cytokine, e.g. interleukin 2 (IL-2), conjugated to antibody against tumour neovasculature antigen, e.g. tenascin-C, for use in combination therapy with chemotherapeutic agent such as temozolomide. EP0229998B1 describes a fusion protein having an IL-2 portion and a desired protein, which has the feature that it contains a C- or N-terminal portion which essentially corresponds to the amino acid sequence of IL-2 but which is not biologically active, excepting fusion proteins whose IL-2 portion corresponds essentially at least to the first 100 amino acids of IL-2.

The interferon alpha (IFN-a) proteins are produced by leukocytes. They are mainly involved in innate immune response against viral infection. They come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17,

IFNA21. These genes for these IFN-a molecules are found together in a cluster on chromosome 9. U.S. Patent US7611700B2 describes protease resistant modified IFN-a polypeptides.

Lee JH, et al. described KISS1 gene: "KiSS-1, a Novel Human Malignant Melanoma Metastasis-Suppressor Gene", Journal of the National Cancer Institute, Vol. 88(23): 1731-1737 (1996). At least four functional peptide products have been reported for KISS1 gene: kisspeptin-54 (also known as metastin), kisspeptin-14, kisspeptin-13, and kisspeptin-10, with 54, 14, 13, and 10 amino acid residues, respectively (Kotani M et al. J. Biol. Chem. Vol. 276(37):34631-34636 (2001); Ohtaki T. et al., Nature Vol. 411(6837):613-671 (2001)). They all share the same 10 amino acids. But they vary in length at the N-terminus. Kisspeptins are amidated at the C-terminus. They have been shown to be functional ligand of KISS1R, a G protein-coupled receptor (US 6699965 Bl). The KISS1/KISS1R is involved in the development of reproductive system, including the onset of puberty (Popa SM et al, Annu. Rev. Physiol. Vol 70:213-38 (2008)). Kisspeptins have been reported to suppress tumor metastasis (Lee JH and Welch DR, Int. J. Cancer. Vol. 71(6): 1035-1044 (1997)). Metastin derivatives also have been reported to have excellent biological activities (a cancer metastasis suppressing activity, a cancer growth suppressing activity, etc.) (for details, see US 6800611 B2, US 7625869 B2, US 8361968 B2, US 8592379 B2). US7112662B2 describes anti-metastin antibodies and their methods of use, thus metastin or its derivative can be quantified specifically with a high sensitivity.

Fusion proteins or chimeric proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. U.S. Patent US8076288B2 describes hybrid polypeptides having glucose lowering activity. US7569384B2 discloses albumin fusion proteins.

Summary of Invention

The present invention relates to a composition of fusion molecules comprising a cytokine moiety and a targeting moiety. The cytokine moiety comprises one or more cytokines. The targeting moiety comprises one or more ligands. Each ligand is a peptide, a protein, or a small molecule that recognizes a receptor on the target, such as but not limited to cells or viral particles. The targeting moiety targets the fusion molecules to the site of the targets and the cytokine moiety exerts its function at the site of the target, thereby resulting in enhanced activity of the cytokine.

In one embodiment of the invention, the cytokine is a fragment or variation of interleukin-2. In another embodiment, the chemokine is GM-CSF, or TNFalpha, or IFNalpha, or IFNbeta, or IFNgamma.

In some embodiments the ligand moiety of the fusion protein comprises at least a fragment or variation of kisspeptins. The kisspeptin moiety targets the fusion molecules to the site of the targets and the cytokine moiety stimulates the proliferation of immune cells at the site of the target, thereby result in enhanced anti-target immune response. In another embodiment, the ligand moiety comprises at least a fragment or variation of endothelin-1, endothelin-2, or endothelin-3 and the cytokine moiety comprises at least a fragment or variation of interleukin-2, or TNF alpha, or IFNalpha, INFbeta, or INFgamma.

Disclosed in this invention is also method of use of the cytokine-ligand fusion to treat human diseases. In some aspects of the invention, tumor cells are the targets and the human disease is a human cancer, such as but not limited to metastatic renal cell carcinoma, or metastatic melanoma. In another embodiment, virus-infected cells are the targets. In yet another embodiment, viral particles are the targets. The composition is used to treat a disease or diseases caused by viral infection.

These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

Brief Description of Drawings

Figure 1 is a schematic diagram of an example of cytokine-ligand fusion molecules of the present invention.

Detailed Description of the Invention

The present invention relates to fusion molecules comprising a functional moiety and a targeting moiety. The functional moiety comprises one or more cytokines. The targeting moiety comprises one or more ligands of receptors expressed on, or attached to the surface of the targets. The ligand is a peptide, or protein, or a small molecule. The targets include but not limited to tumor cells, virus-infected cells, or viral particles. The ligand-cytokine fusion protein provides an enhanced immune response. The targeting moiety concentrates the fusion protein to the site of the targets and the function moiety exerts its function at the site of the targets, thereby resulting in enhanced anti-target activity.

In one embodiment of the invention, the cytokine is a fragment or variation of an interleukins, said interleukins being selected from the group consisting of IL2; IL15; IL4; IL13; IL7; IL9; IL21; IL3; IL5; GM-CSF; IL6; IL11; IL27; IL30; IL31 (+non IL OSM, LIF, CNTF, CTF1); IL12; IL23; IL27; IL35; IL14; IL16; IL32; IL34; IL10/IL22; IL19; IL20; IL24; IL26; IL28A; IL28B; IL29; ILIA; IL1B/IL1F2; lRa/ILlF3; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1F10; IL1F11; IL1G; IL17A; IL17B; and IL25.

In another embodiment, said IL2 comprises at least a fragment or variation of amino acid sequence of

APTSSSTK TQLQLEHLLLDLQMILNGI NYK PKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSK FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT (SEQ ID NO:l). In another embodiment, the cytokine comprises at least one member of TNF family, said TNF family including TNFA/TNFSF2; Lymphotoxin (TNFB/LTA, TNFC/LTB); TNFSF4; TNFSF5/CD40LG; TNFSF6; TNFSF7; TNFSF8; TNFSF9; TNFSFIO; TNFSFl l; TNFSF13B; and EDA.

In another embodiment, said TNFA comprises at least a fragment or variation of amino acid sequence of

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYL GGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO:3).

In another embodiment, the cytokine comprises at least a fragment or variation of an interferon, said interferon including IFNAl; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNA8; IFNAIO; IFNA13; IFNA14; IFNA16; IFNA17; IFNA21; IFNB1; IFNK; IFNW1; and IFNG

In another embodiment, said IFNAl comprises at least a fragment or variation of amino acid sequence of

MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFG FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE ACVMQEERVGETPLMNADSILAVK YFRRITLYLTEK YSPCAWEVVRAEIMRSLSLSTN LQERLRRKE(SEQ ID NO:5).

In another embodiment, said IFNA2 comprises at least a fragment or variation of amino acid sequence of

MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFG FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEA CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNL QESLRSKE(SEQ ID NO:6).

In another embodiment, said IFNB1 comprises at least a fragment or variation of amino acid sequence of

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7).

In another embodiment, said IFNG comprises at least a fragment or variation of amino acid sequence of

MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLK YFNAGHSDVADNGTLFLGILK WK EESDRKIMQSQIVSFYFKLFK FKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO:8).

In another embodiment, said Endothelin-3 comprises at least a fragment or variation of amino acid sequence of CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9)

In another embodiment, the cytokine comprises at least one chemokine, selected from the group consisting of CCL1; CCL2/MCP-1; CCL3/MIP-la; CCL4/MIP-1 P; CCL5/RANTES; CCL6; CCL7; CCL8; CCL9; CCL11; CCL12; CCL13; CCL14; CCL15; CCL16; CCL17; CCL18; CCL19; CCL20; CCL21; CCL22; CCL23; CCL24; CCL25; CCL26; CCL27; CCL28; CXCL1/KC; CXCL2; CXCL3, CXCL4; CXCL5; CXCL6; CXCL7; CXCL8/IL8; CXCL9; CXCL10; CXCL11; CXCL12; CXCL13; CXCL14; CXCL15; CXCL16; CXCL17; CX3CL1; XCL1; and XCL2.

In another embodiment, the cytokine comprises at least one chemokine, selected from the group consisting of KITLG; CSF; and SPP1.

In another embodiment, the chemokine is GM-CSF, or TNF alpha, or IFNalpha, or IFNbeta, or IFNgamma.

In another embodiment, the cytokine comprises at least a fragment or variation of a growth factor, selected from the group consisting of Adrenomedullin (AM); Angiopoietin (Ang); Autocrine motility factor; Bone morphogenetic proteins (BMPs); Brain-derived neurotrophic factor (BDNF); Epidermal growth factor (EGF); Erythropoietin (EPO); Fibroblast growth factor (FGF); Glial cell line-derived neurotrophic factor (GDNF); Granulocyte colony-stimulating factor (G-CSF); Granulocyte macrophage colony-stimulating factor (GM-CSF); Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF); Hepatoma-derived growth factor (HDGF); Insulin-like growth factor (IGF); Migration-stimulating factor; Myostatin (GDF-8); Nerve growth factor (NGF) and other neurotrophins; Platelet-derived growth factor (PDGF); Thrombopoietin (TPO); Transforming growth factor alpha(TGF-a); Transforming growth factor beta(TGF-P); Tumor necrosis factor-alpha(TNF-a); Vascular endothelial growth factor (VEGF); Wnt Signaling Pathway; placental growth factor (P1GF).

In another embodiment, the ligand moiety comprises at least a fragment or variation of endothelin-1, endothelin-2, or endothelin-3 and the cytokine moiety comprises at least a fragment or variation of interleukin-2, or TNF alpha, or IFNalpha, INFbeta, or INFgamma.

In another embodiment, the ligand comprises a fragment or variation of peptide or protein that binds to one or more of the receptors selected from the group consisting of: receptors expressed on hepatocytes before or after infection by a hepatitis virus (HAV, or HBV, or HCV); hepatitis B virus surface antigen (HBVsAg); gpl20 of human immunodeficiency virus (HIV); gp41 of human immunodeficiency virus (HIV); or hemagglutinin of human influenza virus; peplomer protein E2 of SARS-coronavirus; the spike protein of SARS-coronavirus; hemagglutinin-esterase glycoprotein of SARS-coronavirus.

In another embodiment, the ligand comprises a fragment or variation of peptide or protein that binds to one or more of the receptors selected from the group consisting of: BMPR1B (bone morphogenetic protein receptor-type IB); CDH3 (cadherin-3, CDHP); CDH19 (cadherin-19); CDH2 (neural cadherin, NCAD); SLC7A5 (LATl); STEAPl (six transmembrane epithelial antigen of prostate); MUC16 (CA125); KISS1R (GPR54); CLDN3 (Claudin-3); CLDN4 (Claudin-4); CLDN18 (Claudin-18); MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); SLC34A2

(NAPI-3B, NPTIIb, Napi3b solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); IL13RA2 (IL13 receptor alpha 2); PSMA prostate specific membrane antigen); Se2ma 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B); TMEFF1 (Tomoregulin-1); PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); ETBR (Endothelin type B receptor); MSG783 (RNF124, hypothetical protein FLJ20315); CACNA1A (CAV2.1), STEAP2 (PCANAP1, STAMP1, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein); TMEFF2 (Tomoregulin-2); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); OR52E1 (olfactory receptor, family 52, subfamily E, member 1); OR52E2 (olfactory receptor, family 52, subfamily E, member 2, PSGR); CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor); CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792); CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated beta), B29); FcRH2 (IFGP4, IRTA4, SPAPIA (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C); HER2; NCA; MDP; IL20R.alpha.; Brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3; CD22 (B-cell receptor CD22-B isoform); MET (met proto-oncogene, hepatocyte growth factor receptor); CD79a (CD79A, CD79. alpha., immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, transduces a signal involved in B-cell differentiation); CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); CD72 (B-cell differentiation antigen CD72, Lyb-2); LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); FcRHl (Fc receptor- like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation); IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); and TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin).

In some embodiments the ligand moiety of the fusion protein comprises at least a fragment or variation of kisspeptins. The kisspeptin moiety targets the fusion molecules to the site of the targets and the cytokine moiety stimulates the proliferation of immune cells at the site of the target, thereby result in enhanced anti-target immune response.

In another embodiment, said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF SEQ ID NO:2.

In another embodiment, said cytokine is IL2, comprising at least a fragment or variation of amino acid sequence of

APTSSSTK TQLQLEHLLLDLQMILNGI NYK PKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSK FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT(SEQ ID NO:l), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SEQ ID NO:2).

In another embodiment, said cytokine is TNFA, comprising at least a fragment or variation of amino acid sequence of

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYL GGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO:3), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SEQ ID NO:2).

In another embodiment, said cytokine is IFNA1, comprising at least a fragment or variation of amino acid sequence of

MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFG FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE ACVMQEERVGETPLMNADSILAVK YFRRITLYLTEK YSPCAWEVVRAEIMRSLSLSTN LQERLRRKE(SEQ ID NO:5), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SEQ ID NO:2).

In another embodiment, said cytokine is IFNA2, comprising at least a fragment or variation of amino acid sequence of

MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFG FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEA CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNL QESLRSKE(SEQ ID NO:6), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SEQ ID NO:2).

In another embodiment, said cytokine is IFNB1, comprising at least a fragment or variation of amino acid sequence of

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SEQ ID NO:2).

In another embodiment, said cytokine is IFNG, comprising at least a fragment or variation of amino acid sequence of

MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLK YFNAGHSDVADNGTLFLGILK WK EESDRKIMQSQIVSFYFKLFK FKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO: 8), and said ligand comprises a variation or a fragment of kisspeptin with amino acid sequence of

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF(SE Q ID NO:2).

In another embodiment, said cytokine is IL2, omprising at least a fragment or variation of amino acid sequence of

APTSSSTK TQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFC QSIISTLT(SEQ ID NO: l), and said ligand comprises a variation or a fragment of Endothelin-3 with amino acid sequence of

CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9).

In some embodiments of the invention, the targets are tumor cells. In some aspects of the invention, the targeting moiety of the fusion protein comprises at least a fragment or variation of a ligand of a receptor expressed on, or attached to the surface of the tumor cells.

In one preferred embodiment, the fusion molecule comprises at least a fragment or variation of IL-2 and at least a fragment or variation of kisspeptin, or a ligand of KISS 1R.

In another preferred embodiment, the fusion molecule comprises at least a fragment or variation of IL-2 and at least a fragment or variation of a ligand of endothelin receptor A or endothelin receptor B, such as but not limited to endothelin-1, or endothelin-2, or endothelin-3. Yet in another embodiment, the ligand comprises at least a fragment or variation of CD27, or a ligand of CD70. In another aspect of the invention, the function moiety comprises at least a fragment or variation of interleukin-2. Disclosed in this invention is also method of use of the ligand-cytokine fusion to treat human cancers.

In another embodiment, the targets are virus-infected cells or virus particles. The targeting moiety of the fusion protein comprises at least a fragment or variation of a ligand of a receptor expressed on, or attached to the surface of the cells that virus are to infected, such as but not limited to, receptors expressed on hepatocytes before or after infection by a hepatitis virus (HAV, or HBV, or

HCV). In another embodiments, the targeting moiety comprises at least a fragment or a variation of a surface antigen or coat protein of viral particles, such as but not limited to hepatitis B virus surface antigen (HBVsAg), or gpl20 of human immunodeficiency virus (HIV), or gp41 of human immunodeficiency virus (HIV), or hemagglutinin of human influenza virus, or peplomer protein E2 of SARS-coronavirus, or the spike protein of SARS-coronavirus, or hemagglutinin-esterase glycoprotein of SARS-coronavirus. The functional moiety is fragment or variation of interferon alpha, or interferon beta, or interferon gamma, or interleukin-2. Disclosed in this invention is also method of use of the ligand-cytokine fusion to treat human viral diseases.

An exemplary diagrammatic depiction of the present invention is provided in Figures 1, which shows a composition comprising one targeting moiety and one cytokine moieties. These functional moieties can be fused directly together or with various linkers.

The variations of the compositions are discussed further below.

In one embodiment, the fusion molecule comprises one targeting moiety, a peptide or a protein ligand of a receptor on tumor cells and one cytokine moiety, as illustrated in Figure 1.

In another embodiment, one targeting moiety is fused to more than one cytokine moieties. The cytokine moieties can be identical, or different. The targeting moiety can be at the N-terminus, at the C-terminus or in between the cytokine moieties.

In another embodiment, more than one targeting moieties are fused to one cytokine. The targeting moieties can be identical, or different. The cytokine moiety can be at the N-terminus, at the C-terminus or in between the targeting moieties.

Yet in another embodiment, more than one targeting moieties are fused to more than one moieties of cytokines. The targeting moieties can be identical or different. The cytokine moieties also can be identical or different. The cytokine moieties can be at the N-terminus, at the C-terminus or in between the targeting moieties. The cytokine moieties can be together or dispersed among targeting moieties. The targeting moieties can also be together or dispersed among cytokine moieties.

The cytokine-receptor ligand fusion could be constructed with yet another moiety, such as antibody, or human albumin or other proteins that can bring longer half life to the fusion protein.

The present invention thus in one aspect provides a composition of a fusion molecule comprising a targeting moiety and a cytokine moiety. In another aspect, the current invention provides methods of using such composition for treating diseases. Also provided are kits, unit dosages, pharmaceutical compositions, medicines, and article of manufacture that are useful for the uses described herein.

Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Compositions comprising function moiety and targeting moiety This fusion molecule comprises at least one cytokine moiety, such as a biologically active lymphokine and at least one targeting moiety, such as a peptide or protein ligand of receptors expressed on target cells or target viral particles. The ligand-cytokine fusion protein provides an amplified immune response. The ligand moiety targets the fusion protein to the site of the target cells and the cytokine portion stimulates the proliferation of immune cells at the site of the target cells, thereby amplifying the anti-target immune response.

For example, in some embodiments, the targeting moiety comprises a peptide ligand to a receptor expressed on the target. The binding affinity of the targeting moiety for the receptor on the target is in the fJVI range to μΜ range (including, for example, about 10 fJVI to about 1 pM, about 1 pM to about 10 pM, about 100 pM to about 10 nM, about 10 nM to about 1 μΜ, about 1 μΜ to about 100 μΜ). In some embodiments, the binding affinity of the targeting moiety to the receptor is about 10 ^"14 M to about 10 ^"5 M.

Thus in some embodiments, there is provided a composition comprising a fragment or variation of the product of the KISS1 gene (such as but not limited to kisspeptins or metastin) as the targeting moiety, and the interleukin-2 as the cytokine moiety. In some embodiments, the targeting moiety comprises a fragment or variation of endothelin-3, and the cytokine moiety comprises a fragment or variation of interleukin-2.

In some aspects of the invention, the fusion molecule is produced by the skilled in art in various expression systems, including but not limited to E. coli, yeast, mammalian cells and purified by skilled in the art, employing various technology, including but not limited to column chromatography.

In some other embodiments, the targeting moiety comprises a protein ligand to a receptor expressed on the target. The binding affinity of the targeting moiety for the receptor on the target is in the fJVI range to μΜ range (including, for example, about 10 fJVI to about 1 pM, about 1 pM to about 100 pM, about 100 pM to about 10 nM, about 10 nM to about 1 μΜ, about 1 μΜ to about 100 μΜ). In some embodiments, the binding affinity of the targeting moiety to the receptor is about 10 ^"14 M to about 10 ^"5 M.

For example, in some embodiments, there is provided a composition comprising at least a fragment or variation of CD27, a ligand to CD70 on renal cell carcinoma, and the interleukin-2 as the cytokine moiety.

In some aspects of the invention, the fusion molecule is produced by the skilled in art in various expression system, including but not limited to E. coli, yeast, mammalian cells and purified by skilled in art, employing various technology, including but not limited to column chromatography

For example, in yet some other embodiments, the targeting moiety comprises a small molecule ligand to a receptor expressed on the target. The binding affinity of the targeting moiety for the receptor on the target is in the fM range to μΜ range (including, for example, about 10 fM to about 1 pM, about lpM to about 100 pM, about 100 pM to about 10 nM, about 10 nM to about 1 μΜ, about 1 μΜ to about 100 μΜ). In some embodiments, the binding affinity of the targeting moiety to the receptor is about 10 ^"1"* M to about 10 M.

In some aspects of the invention, the fusion molecule is produced by the skilled in the art in various expression systems, including but not limited to E. coli, yeast, mammalian cells and purified by skilled in the art, employing various technology, including but not limited to column chromatography.

The compositions described herein can be provided in a pharmaceutical composition. The pharmaceutical composition can comprise a pharmaceutically acceptable carrier. In some embodiments, the composition is contained in a vial (such as a sealed vial) or a delivery device.

The pharmaceutical composition in some embodiments comprises a buffer substance, such as, but not limited to, a phosphate, for example sodium phosphate, glycine or glycylglycine buffer, an isotonicity agent, such as sodium chloride or glycerol, and phenol and/or m-cresol as a preservative. In some embodiments, isotonic agents such as mannitol or sorbitol can be added. For example, in some embodiments, the pharmaceutical composition comprises chloride ions in a concentration of about 5 to about 150 mM.

In some embodiments, the pharmaceutical composition is a liquid composition, and the amount of the fusion molecule in the composition is in the range from about 0.001 to about 15 mM. Phenolic compounds like phenol or m-cresol or mixtures thereof can also be present, for example at a total concentration of from about 5 to about 50 mM, and optionally chloride ions in a concentration of from about 10 mM to about 200 mM.

In some embodiments, the pH in the pharmaceutical composition is in the range of about 2.0 to about 10.0, including for example about 2.0 to about 3.0, about 3.0 to about 4.0, about 4.0 to about 5.0, about 5.0 to about 6.0, about 6.0 to about 7.0, about 7.0 to about 8.0, about 8.0 to about 9.0, about 9.0 to about 10.0.

Methods of using the compositions

The compositions described herein can be used for treating diseases. For example, the preferred embodiment IL2-kisspeptin fusion can be used to treat such as but not limited to, metastatic renal cell carcinoma, or metastatic melanoma.

The administration of the composition can be carried out alone or in conjunction with other agents suitable for the same purpose. For example, the composition can be used in combination with other therapeutics, such as chemotherapy or other targeted therapeutics.

The composition can be delivered by any suitable route. In some embodiments, the composition is delivered by infusion intravenously, intraperitoneally, subcutaneously, intramuscularly, by reservoir/pumping devices, by skin patches, or by inhalation.

The composition in some embodiments can be administered once a day or less frequently. For example, in some embodiments, the composition is administered once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, monthly, bimonthly, every three months, or every six months.

The optimal dose level of the effective molecule will depend on a variety of factors including the age, body weight, physical condition of the patients, on a possible combination with other drugs, and on the severity of the case of disease. The specific dosage can be determined by those skilled in the art in a similar way as for known cytokine molecule compositions.

Kits, unit dosages, and articles of manufacture

The present invention further provides kits, unit dosages, and articles of manufacture comprising compositions described herein. The kits, unit dosages, and article of manufacture may comprises, for example, vials (such as sealed vials) comprising the compositions described herein.

Example 1. Expression of IL2-kisspeptin fusions in Mammalian cells

This example describes a method of preparing an IL2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO:2a) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence APTSSSTK TQLQLEHLLLDLQMILNGI NYK PKLTRMLTFKFYMPKKATELKHLQCLEEEL KPLEEVLNLAQSK FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII STLT(SEQ ID NO: l). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 2. Expression of IL2-kisspeptin fusions in E. coli

This example describes a method of preparing an IL2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID

NO:2a) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ CLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSII

STLT(SEQ ID NO: 1). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor.

To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 3. Expression of IL2-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing an IL2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence APTSSSTK TQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: l). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 4. Expression of IL2-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing an IL2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO:2a) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: l). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 5. Expression of TNFA-kisspeptin fusions in Mammalian cells

This example describes a method of preparing a TNFA-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID

NO :2a) is fused to the C-terminus of TNFA with amino acid sequence

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYL IYSQV

LFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIY LGGVFQLE

KGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO:3). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a

CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an

RF-amide at the C-terminus of the fusion protein.

Example 6. Expression of TNFA-kisspeptin fusions in E. coli

This example describes a method of preparing a TNFA-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID NO :2a) is fused to the C-terminus of TNFA with amino acid sequence VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QV LFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLE KGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO:3). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG The fusion protein is purified by the skilled in the art by column chromatography.

Example 7. Expression of TNFA-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing a TNFA -kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID

NO :2a) is fused to the C-terminus of TNFA with amino acid sequence

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYL IYSQV

LFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIY LGGVFQLE

KGDRLSAETNRPDYLDFAESGQVYFGIIAL(SEQ ID NO:3). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently trans feet the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 8. Expression of TNFA-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing a TNFA-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of TNFA with amino acid sequence VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QV LFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLE KGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO:3). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG The fusion protein is purified by the skilled in the art by column chromatography.

Example 9. Expression of IFNAl-kisspeptin fusions in Mammalian cells

This example describes a method of preparing an IFNAl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNA1 with amino acid sequence MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFG FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE ACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTN LQERLRRKE(SEQ ID NO:5). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 10. Expression of IFNAl-kisspeptin fusions in E. coli This example describes a method of preparing an IFNAl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID NO :2a) is fused to the C-terminus of IFNA1 with amino acid sequence MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNPvRTLMLLAQMSRISPSSCLMDRHDF G FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE ACVMQEERVGETPLMNADSILAVK YFRRITLYLTEK YSPCAWEVVRAEIMRSLSLSTN LQERLRRKE(SEQ ID NO:5). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 11. Expression of IFNAl-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing an IFNAl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNA1 with amino acid sequence MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFG FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE ACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTN LQERLRRKE(SEQ ID NO:5). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 12. Expression of IFNAl-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing an IFNAl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID

NO :2a) is fused to the C-terminus of IFNA1 with amino acid sequence

MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRH DFG FPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLE

ACVMQEERVGETPLMNADSILAVK YFRRITLYLTEK YSPCAWEVVRAEIMRSLSLSTN

LQERLRRKE(SEQ ID NO:5). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the

C-terminus of the fusion protein. The expression of the fusion is induced by IPTG The fusion protein is purified by the skilled in the art by column chromatography.

Example 13. Expression of IFNA2-kisspeptin fusions in Mammalian cells

This example describes a method of preparing an IFNA2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNA2 with amino acid sequence MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFG FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEA CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNL QESLRSKE(SEQ ID NO:6). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 14. Expression of IFNA2-kisspeptin fusions in E. coli

This example describes a method of preparing an IFNA2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID

NO :2a) is fused to the C-terminus of IFNA2 with amino acid sequence

MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRH DFG

FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLND LEA

CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLS TNL

QESLRSKE(SEQ ID NO:6). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 15. Expression of IFNA2-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing an IFNA2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNA2 with amino acid sequence MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFG FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEA CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNL QESLRSKE(SEQ ID NO:6). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 16. Expression of IFNA2-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing an IFNA2-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID

NO :2a) is fused to the C-terminus of IFNA2 with amino acid sequence

MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRH DFG

FPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLND LEA

CVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLS TNL

QESLRSKE(SEQ ID NO:6). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the

C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography. Example 17. Expression of IFNBl-kisspeptin fusions in Mammalian cells

This example describes a method of preparing an IFNBl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNB1 with amino acid sequence MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 18. Expression of IFNBl-kisspeptin fusions in E. coli

This example describes a method of preparing an IFNBl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID NO :2a) is fused to the C-terminus of IFNB1 with amino acid sequence MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQTNHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 19. Expression of IFNBl-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing an IFNBl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNB1 with amino acid sequence

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRM NFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 20. Expression of IFNBl-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing an IFNBl-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNB1 with amino acid sequence MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN(SEQ ID NO:7). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG The fusion protein is purified by the skilled in the art by column chromatography.

Example 21. Expression of IFNG-kisspeptin fusions in Mammalian cells

This example describes a method of preparing an IFNG-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID

NO :2a) is fused to the C-terminus of IFNG with amino acid sequence

MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILK NWK

EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEK LTN

YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO: 8). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently trans feet the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No. 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 22. Expression of IFNG-kisspeptin fusions in E. coli

This example describes a method of preparing an IFNG-kisspeptin(such as, but not limited to, kisspeptin-54) fusion. Kisspeptin-54 with amino acid sequence

GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG(S EQ ID NO :2a) is fused to the C-terminus of IFNG with amino acid sequence MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLK YFNAGHSDVADNGTLFLGILKNWK EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO: 8). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG The fusion protein is purified by the skilled in the art by column chromatography.

Example 23. Expression of IFNG-kisspeptin fusion with a linker in Mammalian cells

This example describes a method of preparing an IFNG-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNG with amino acid sequence MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO:8). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein. Example 24. Expression of IFNG-kisspeptin fusion with a linker in E. coli

This example describes a method of preparing an IFNG-kisspeptin(such as, but not limited to, kisspeptin-54) fusion with a linker. Kisspeptin-54 with amino acid sequence GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFG (SEQ ID NO :2a) is fused to the C-terminus of IFNG with amino acid sequence MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLK YFNAGHSDVADNGTLFLGILKNWK EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO:8). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 25. Expression of IL2-endthelin fusions in Mammalian cells

This example describes a method of preparing an IL2-endothelin(such as, but not limited to, Endothelin-3) fusion. Endothelin-3 with amino acid sequence CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence APTSSSTK TQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: l). This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography.

Example 26. Expression of IL2-endothelin fusions in E. coli

This example describes a method of preparing an IL2-endothelin(such as, but not limited to, Endothelin-3) fusion. Endothelin-3 with amino acid sequence CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: 1). This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 27. Expression of IL2-endothelin fusion with a linker in Mammalian cells

This example describes a method of preparing an IL2-endothelin (such as, but not limited to, Endothelin-3) fusion with a linker. Endothelin-3 with amino acid sequence CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence

APTSSSTK TQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: l). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning, with or without a linker between the two moieties and expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The fusion protein is expressed by transiently transfect the CHO or HEK293 cells with an expression vector containing a CMV promoter. The fusion protein is purified by the skilled in the art by column chromatography. In mammalian cells, the last amino acid G in (SEQ ID No: 2a) is converted to an amide, resulting in an RF-amide at the C-terminus of the fusion protein.

Example 28. Expression of IL2-endothelin fusion with a linker in E. coli

This example describes a method of preparing an IL2-endothelin(such as, but not limited to, Endothelin-3) fusion with a linker. Endothelin-3 with amino acid sequence CTCFTYKDKECVYYCHLDIIW (SEQ ID NO:9) is fused to the C-terminus of IL2 (desAlal and C125S) with amino acid sequence

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ CLEEEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSII STLT(SEQ ID NO: l). In between the two moieties, a linker with the sequence of, such as but not limited to, GGGSGGGS (SEQ ID NO:4) is inserted. This fusion will be made by the skilled in the art by DNA cloning and expressed in E. coli. The expression construct contains a T3 or T7 promoter under the control of LacZ repressor. To facilitate amidation of the fusion protein at the C-terminus, a bi-functional enzyme called peptidylglycine alpha-amidating monooxygenase will be co-expressed in the E. coli. This enzyme converts the last amino acid Gly to amide, resulting in RF-amide at the C-terminus of the fusion protein. The expression of the fusion is induced by IPTG. The fusion protein is purified by the skilled in the art by column chromatography.

Example 29. IL2 Activity of IL2-Kisspeptin fusion

IL2-Kisspeptin (such as, but not limited to, kisspeptin-54) was cloned into pCDNA3.1, and transiently transfected into HEK293 cells, affinity-purified with cleavable tag and tested for their IL2 activity in maintaining CTLL-2 cells. CTLL-2 cells were cultured in DMEM medium, supplemented with heat-inactivated fetal calf serum, and serially diluted IL2, IL2-kisspeptin, or kisspeptin. CTLL-2 cell numbers were measured by MTT assay.

Table 1. IL2 Activity measured by MTT assay

*: N/A: No activity detected

Example 30. Potency ofIL2-Kisspeptin fusion, and kisspeptin to activate KISS1R

Stable cell line CHO-K1/KISS1R and the parental CHO-K1 cells were transfected with Galphal6. Calcium mobilization was measured with calcium-sensitive fluorescent dyes, as previously described (Kotani et al, (2001) Journal of Biological Chemistry, Vol 276: 34631).

Table 2. Potency measured with calcium-sensitive fluorescent dyes

*: N/A: No activity detected

Example 31. IL2-Kisspeptin fusion is superior to IL2 in killing kisspeptin expressing tumor cells

The cytotoxicity of the fusion proteins were measured using the pair of Daudi lymphoma cells and Daudi/KISSIR (Daudi stably expressing KISS1R) cell lines. First, peripheral blood mononuclear cells (PBMCs) were seeded in a 96-well plate. Cells were incubated and allowed to attach to the wells while unattached cells were rinsed off. Cells attached to the wells were used as the effector cells. The targetcells (effector cells: target =15) were incubated with IL2-Kisspeptin fusion proteins, IL2, or Kisspeptins at room temperature, and then added to effector cells. The plates were incubated at 37°C for 40 hours. Supernatant was transferred to another plate and the lactate dehydrogenase released from lysed cells were measured.

Table 3. Ability of killing kisspeptin expressing tumor cells

*: N/A: No activity detected

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Industrial Applicability

The cytokine fusion molecules disclosed by the present invention comprise a cytokine moiety and a targeting moiety. The targeting moiety targets the fusion molecules to the site of the targets and the cytokine moiety exerts its function at the site of the target, thereby resulting in enhanced activity of the cytokine. The cytokine fusion can be used to treat human diseases.

<110> Admark Healthcare, LLC

<120> FUSION PROTEIN MOLECULES AND METHOD OF USE

<130> KHP143110520

<150> US provisional Patent 61802169

<151> 2013-03-15

<160> 10

<170> Patentln version 3.5

<210> 1

<211> 133

<212> PRT

<213> Homo sapiens

<400> 1

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15

Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu 65 70 75 80

Arg Pro Arg Asp Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Cys Gin Ser lie 115 120 125

Ser Thr Leu Thr

130

<210> 2

<211> 54

<212> PRT

<213> Homo sapiens

<400> 2

Gly Thr Ser Leu Ser Pro Pro Pro Glu Ser Ser Gly Ser Pro Gin Gin 1 5 10 15

Pro Gly Leu Ser Ala Pro His Ser Arg Gin lie Pro Ala Pro Gin Gly

20 25 30

Ala Val Leu Val Gin Arg Glu Lys Asp Leu Pro Asn Tyr Asn Trp Asn 35 40 45 Ser Phe Gly Leu Arg Phe

50

<210> 2a

<211> 55

<212> PRT

<213> Homo sapiens

<400> 2a

Gly Thr Ser Leu Ser Pro Pro Pro Glu Ser Ser Gly Ser Pro Gin Gin 1 5 10 15

Pro Gly Leu Ser Ala Pro His Ser Arg Gin lie Pro Ala Pro Gin Gly

20 25 30

Ala Val Leu Val Gin Arg Glu Lys Asp Leu Pro Asn Tyr Asn Trp Asn 35 40 45

Phe Gly Leu Arg Phe Gly

50 55

<210> 3

<211> 157

<212> PRT

<213> Homo sapiens

<400> 3

Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15

Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu Asn Arg Arg

20 25 30

Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gin Leu 35 40 45

Val Val Pro Ser Glu Gly Leu Tyr Leu lie Tyr Ser Gin Val Leu Phe 50 55 60

Lys Gly Gin Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr lie 65 70 75 80

Ser Arg lie Ala Val Ser Tyr Gin Thr Lys Val Asn Leu Leu Ser Ala

85 90 95 lie Lys Ser Pro Cys Gin Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys

100 105 110

Pro Trp Tyr Glu Pro lie Tyr Leu Gly Gly Val Phe Gin Leu Glu Lys 115 120 125

Gly Asp Arg Leu Ser Ala Glu lie Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140

Ala Glu Ser Gly Gin Val Tyr Phe Gly He lie Ala Leu

145 150 155

<210> 5

<211> 189

<212> PRT

<213> Homo sapiens

<400> 5

Met Ala Ser Pro Phe Ala Leu Leu Met Val Leu Val Val Leu Ser Cys 1 5 10 15

Lys Ser Ser Cys Ser Leu Gly Cys Asp Leu Pro Glu Thr His Ser Leu

20 25 30

Asp Asn Arg Arg Thr Leu Met Leu Leu Ala Gin Met Ser Arg lie Ser 35 40 45

Pro Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly Phe Pro Gin Glu 50 55 60

Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Pro Ala lie Ser Val Leu 65 70 75 80

His Glu Leu lie Gin Gin lie Phe Asn Leu Phe Thr Thr Lys Asp Ser

85 90 95

Ser Ala Ala Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu

100 105 110

Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Met Gin Glu Glu Arg 115 120 125

Val Gly Glu Thr Pro Leu Met Asn Ala Asp Ser lie Leu Ala Val Lys 130 135 140

Lys Tyr Phe Arg Arg lie Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser 145 150 155 160

Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg Ser Leu Ser

165 170 175

Leu Ser Thr Asn Leu Gin Glu Arg Leu Arg Arg Lys Glu

180 185

<210> 6

<211> 188

<212> PRT <400> 6

Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15

Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gin Thr His Ser Leu

20 25 30

Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg Lys lie Ser 35 40 45

Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu 50 55 60

Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr lie Pro Val Leu His 65 70 75 80

Glu Met lie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser

85 90 95

Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr

100 105 110

Gin Gin Leu Asn Asp Leu Glu Ala Cys Val lie Gin Gly Val Gly Val 115 120 125

Thr Glu Thr Pro Leu Met Lys Glu Asp Ser lie Leu Ala Val Arg Lys 130 135 140

Tyr Phe Gin Arg lie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro 145 150 155 160

Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg Ser Phe Ser Leu

165 170 175

Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu

180 185

<210> 7

<211> 187

<212> PRT

<213> Homo sapiens

<400> 7

Met Thr Asn Lys Cys Leu Leu Gin lie Ala Leu Leu Leu Cys Phe Ser 1 5 10 15

Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gin Arg

20 25 30

Ser Ser Asn Phe Gin Cys Gin Lys Leu Leu Trp Gin Leu Asn Gly Arg 35 40 45

Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp lie Pro Glu Glu 50 55 60

lie Lys Gin Leu Gin Gin Phe Gin Lys Glu Asp Ala Ala Leu Thr lie 65 70 75 80 Tyr Glu Met Leu Gin Asn lie Phe Ala lie Phe Arg Gin Asp Ser Ser 85 90 95

Ser Thr Gly Trp Asn Glu Thr lie Val Glu Asn Leu Leu Ala Asn Val

100 105 110

Tyr His Gin lie Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125

Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140

Arg Tyr Tyr Gly Arg lie Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160

His Cys Ala Trp Thr lie Val Arg Val Glu lie Leu Arg Asn Phe Tyr

165 170 175

Phe lie Asn Arg Leu Thr Gly Tyr Leu Arg Asn

180 185

<210> 8

<211> 166

<212> PRT

<213> Homo

<400> 8

Met Lys Tyr Thr Ser Tyr lie Leu Ala Phe Gin Leu Cys lie Val Leu 1 5 10 15

Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu

20 25 30

Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn 35 40 45

Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp 50 55 60

Arg Lys lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe 65 70 75 80

Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie

85 90 95

Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg

100 105 110

Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val 115 120 125

Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala Glu Leu Ser 130 135 140

Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg 145 150 155 160

Gly Arg Arg Ala Ser Gin

165 <210> 9

<211> 21

<212> PRT

<213> Homo sapiens

<400> 9

Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His 1 5 10 15

Leu Asp lie lie Trp

20