Fc-CXCL14 FUSION PROTEINS

Abstract

The present invention relates to a fusion protein comprising a CXCL14 sequence linked to an immunoglobulin Fc region sequence, methods for preparation thereof, and methods for using it for treating cancer or metabolic syndrome diseases.

Description

[0001] Fc-CXCL14 FUSION PROTEINS

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to immunological treatment. More specifically, the invention relates to modified chemokines for treating cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Chemokines are a family of small proteins that are crucial to the regulation of immune cell trafficking during homeostasis and inflammation. They act by binding to their receptors, which are seven-transmembrane G protein-coupled receptors.

[0006] CXCL14 is a chemokine that belongs to the CXC subfamily and is also known as breast and kidney-expressed chemokine (BRAK). CXCL14 is mainly expressed in normal tissues, including liver, kidney, and breast tissue. It is known to regulate immune cell trafficking and function in various physiological and pathological conditions.

[0007] CXCL14 is one of the most evolutionarily conserved chemokines, exhibiting remarkable similarity across a wide range of mammals and reptiles

[0008] In cancer, CXCL14 may have a dual role. In certain types of cancer such as hepatocellular carcinoma (HCC), CXCL14 acts as a tumor suppressor, with levels decreasing almost completely and overexpression causing tumor growth arrest. In other types of cancer, CXCL14 levels increase, and it participates in inflammatory processes within tumors which lead to arresting tumor growth.

[0009] Due to the short half-life of chemokines in the blood, which is about half an hour, they cannot be used directly as treatment agents. Accordingly, there is a need for developing a more effective form of CXCL14 that may be used for improving cancer treatments.

[0010] SUMMARY OF INVENTION

[0011] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0012] In some embodiments, there is provided a fusion protein including a CXCL14 sequence linked to an immunoglobulin Fc region sequence.

[0013] In some embodiments, at least one of the CXCL14 sequence and the Fc region sequence is derived from a human sequence. In some embodiments, the CXCL14 sequence includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1. In some embodiments, the CXCL14 sequence has a length of about 50-150, 50-120, 60-120, or 70-110 amino acids.

[0014] In some embodiments, the immunoglobulin Fc region sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to an immunoglobulin Fc region of an immunoglobulin selected from IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. In some embodiments, the immunoglobulin Fc region sequence includes a complete immunoglobulin Fc region sequence In some embodiments, the immunoglobulin Fc region sequence lacks the CHI region of the immunoglobulin Fc region, in order to prevent antibody-dependent cellular cytotoxicity (ADCC In some embodiments, the immunoglobulin Fc region sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 2.

[0015] In some embodiments, the immunoglobulin Fc region sequence is N-terminal to the CXCL14 sequence in the fusion protein. In some embodiments, the immunoglobulin Fc region sequence is C-terminal to the CXCL14 sequence in the fusion protein.

[0016] In some embodiments, the fusion protein further includes a linker between the immunoglobulin Fc region sequence and the CXCL14 sequence. In some embodiments, the linker is a flexible linker rich in glycine, serine and / or threonine. In some embodiments, the linker has a length of about 4-40, 4-35, 4-30, or 5-25 amino acids. In some embodiments, the linker has a sequence at least 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 3.

[0017] In some embodiments, the fusion protein further includes a signal peptide at the N-terminus of the fusion protein.

[0018] In some embodiments, the fusion protein includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 5.

[0019] In some embodiments, the fusion protein is conjugated to an anti-cancer drug.

[0020] In some embodiments, there is provided a nucleic acid molecule including a nucleotide sequence encoding the fusion protein disclosed herein.

[0021] In some embodiments, there is provided a vector including the nucleic acid molecule disclosed herein.

[0022] In some embodiments, there is provided a cell including the nucleic acid molecule disclosed herein or the vector disclosed herein, and / or expressing the fusion protein disclosed herein.

[0023] In some embodiments, there is provided a pharmaceutical composition including the fusion protein disclosed herein, the nucleic acid molecule disclosed herein, the vector disclosed herein, or the cell disclosed herein, and a pharmaceutically acceptable carrier.

[0024] In some embodiments, there is provided the pharmaceutical composition disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0025] In some embodiments, there is provided the fusion protein disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0026] In some embodiments, there is provided the nucleic acid disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0027] In some embodiments, there is provided the vector disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0028] In some embodiments, there is provided the cell disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0029] In some embodiments, there is provided a method of treating a cancer or a metabolic syndrome disease in a subject in need thereof, the method including administering to the subject the pharmaceutical composition disclosed herein.

[0030] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), breast cancer, head and neck cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, and renal cancer.

[0031] In some embodiments, the metabolic syndrome disease is selected from obesity, insulin resistance, type 2 diabetes, hyperglycemia, dyslipidemia, elevated triglycerides, low HDL cholesterol, hypertension, non-alcoholic fatty liver disease (NAFLD), atherosclerosis, polycystic ovary syndrome and sleep apnea.

[0032] In some embodiments, the treating includes administering the pharmaceutical composition disclosed herein in combination with an anti-cancer treatment.

[0033] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0036] Figs. 1A-1B show Fc-CXCL14 isolated from cells lysate and detected by CXCL14 ELISA. Fig. 1A. Fc-CXCL14 cleaned on a Protein A column from transfected cells media. Left - size marker (kb), right - Fc-CXCL14. As indicated by the arrow, the Fc-CXCL14 fusion appears as a dimer of 75 kDa. Fig. IB. shows protein levels, detected by ELISA, of Fc-CXCL14 (left), CXCL14 (middle) and CXCL12 (right). As may be seen, Fc-CXCL14 is detected as CXCL14, while CXCL12 (negative control) is not detected, ns: not significant.

[0037] Figs. 2A-2B show enhanced proliferation of LX-2 Cells induced by CXCL14-Fc compared to CXCL14. X axis, Time (T) in hours (h); Y axis, LX-2 cells confluence (%) Fig. 2A. CXCL14 enhances LX-2 cell proliferation. Fig. 2B. Fc-CXCL14 enhances LX-2 cell proliferation to a greater extent than CXCL14, indicating that Fc-CXCL14 acts as a super agonist. For Figs. 2A-2B: circles (untreated, UT); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml.

[0038] Figs. 3A-3C show decreased proliferation in FLC-4 cells treated with Fc-CXCL14. Fig. 3A. Detection of binding of lOOng Fc-CXCL14 to FLC-4 cells using phycoerythrin (PE)-Conjugated Anti-FC Antibody. US: unstained; UT: untreated; 100 ng: FLC-4 cells treated with lOOng Fc- CXCL14. Increased MFI is seen in cells incubated with Fc-CXCL14 compared to untreated cells. Fig. 3B. FLC-4 cells treated with CXCL14 in different doses shows decreased proliferation of the cells. Fig. 3C. FLC-4 cells treated with different doses of Fc-CXCL14 shows decreased proliferation with a greater effect than cells treated with CXCL14. Figs. 3B-3C: UT: Untreated (circles); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml. X axis, Time (T) in hours (h); Y axis, FLC-4 cells confluence (%).

[0039] Figs. 4A-4C show decreased proliferation in Huh7 cells treated with Fc-CXCL14. Fig. 4A. Detection of binding of lOOng Fc-CXCL14 to Huh7 cells using PE-Conjugated Anti-FC Antibody. US: unstained; UT: untreated; 100 ng: Huh7 cells treated with lOOng Fc-CXCL14. Increased MFI is seen in cells incubated with Fc-CXCL14 compared to untreated cells. Fig. 4B. Huh7 cells treated with CXCL14 in different doses shows decreased proliferation of the cells. Fig. 4C. Huh7 cells treated with different doses of Fc-CXCL14 shows decreased proliferation with a greater effect than cells treated with CXCL14. Figs. 4B-4C: circles (untreated, UT); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml. X axis, Time (T) in hours (h); Y axis, Huh7 cells confluence (%).

[0040] Figs. 5A-5C show decreased proliferation in MDA-231 breast cancer cells treated with Fc- CXCL14. Fig. 5A. Detection of binding of lOOng Fc-CXCL14 to MDA-231 cells using PE- Conjugated Anti-FC Antibody. US: unstained; UT: untreated; 100 ng: MDA-231 cells treated with lOOng Fc-CXCL14. Increased MFI is seen in cells incubated with Fc-CXCL14 compared to untreated cells. Fig. 5B. MDA-231 cells treated with CXCL14 in different doses shows decreased proliferation of the cells. Fig. 5C. MDA-231 cells treated with different doses of Fc-CXCL14 shows decreased proliferation with a greater effect than cells treated with CXCL14. Figs. 5B-5C: circles (untreated, UT); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml. X axis, Time (T) in hours (h); Y axis, MDA-231 cells confluence (%). Figs. 6A-6B show decreased proliferation in MDA468 breast cancer cells treated with Fc- CXCL14. Fig. 6A. MDA468 cells treated with CXCL14 at different doses show inhibition of cell proliferation at the highest dose. Fig. 6B. MDA468 cells treated with different doses of Fc- CXCL14 show decreased cell proliferation in a dose dependent manner. Circles (untreated, UT); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml. X axis, Time (T) in hours (h); Y axis, MDA468 cells confluence (%).

[0041] Figs. 7A-7B show decreased proliferation in DLD1 colon cancer cells treated with Fc- CXCL14. Fig. 7A. Different doses of CXCL14 have no effect on the proliferation of the cells. Fig. 7B. Different doses of Fc-CXCL14 result in decreased proliferation of the cell in a dose dependent manner. Circles (untreated, UT); squares: 1 ng / ml; triangles: 10 ng / ml; diamonds: 100 ng / ml; asterisks: 1000 ng / ml. X axis, Time (T) in hours (h); Y axis, DLD1 cells confluence (%).

[0042] Fig. 8 shows that CXCL14 binding to LX-2 Stellate Cells is independent of CXCR4 interaction. Fc-CXCL14 binding to LX-2 cells is detected by using PE-Conjugated Anti-FC Antibody. SDF-1 (CXCL12), the CXCR4 ligand, is used to block CXCR4 (right-most column). As shown, Fc-CXCL14 binding to the cells is not blocked, indicating that it is not via CXCR4. *: P<0.05. UT: untreated cells.

[0043] Fig. 9 shows that the ligands for chemokine receptors CCR2 and CCR5 inhibit the binding of Fc-CXCL14 to the stellate LX-2 cell line. LX-2 cells pre incubated with the ligands of CCR5 and CCR2. MIPla, MIPlb, and MCP-1, or combination of those chemokines showed lower binding of Fc-CXCL14 as detected by anti-Fc antibody conjugated to PE, indicating one of those chemokine receptors is the receptor of CXCL14. ***: P< 0.001. UT: untreated cells. US: unstained cells.

[0044] Fig. 10 shows that ligands for the chemokine receptors CCR2, CCR5 and CXCR4 inhibit the binding of Fc-CXCL14 to monocytic THP-1 cancer cell line. THP-1 cells pre incubated with the ligands of CCR2, CCR5, CXCR4, RANTES, MIPla, MIPlb, MCP-1 and SDF-1 or combination of those chemokines showed less binding of Fc-CXCL14 detected by anti-Fc antibody conjugated to PE, reinforce the hypothesis that one of those chemokine receptors is the receptor of CXCL14. ***: P< 0.001. UT: untreated cells. US: unstained cells.

[0045] Figs. 11A-11C shows results of Fc-CXCL14 safety experiment. Fig. 11A shows the levels of Fc-CXCL14 in NSG mice blood 4 hours, 24 hours, 3 days, 6 days and 10 days following an i.v injection of lOOug of Fc-CXCL14. X axis, T. p-i. (Time post-injection) 100 ug / mouse of CXCL14-FC; Y axis, IgG-Fc ug / ml. The levels of the fusion protein remain high compared to the control group. Negative values indicate no protein detected (the measured optical density (OD) was below the lowest value of the standard curve). Fig. 11B shows no significant changes in weight (W, mg) of organs between the two groups. Fig. 11C shows blood test results 13 days post injection, which generally shows no significant changes between controls and mice injected with Fc-CXCL14. Parameters measured: WBC: white blood cells, RBC: red blood cells, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0048] CXCL14 is a chemokine that belongs to the CXC subfamily and is also known as breast and kidney-expressed chemokine (BRAK). CXCL14 is mainly expressed in normal tissues, including liver, kidney, and breast tissue. It is known to regulate immune cell trafficking and function in various physiological and pathological conditions.

[0049] Recent studies have shown that CXCL14 has antitumor activity in hepatocellular carcinoma (HCC) in vitro and in vivo, and that CXCL14 expression is suppressed in HCC. Moreover, CXCL14 has been shown to attenuate triple-negative breast cancer progression by regulating immune profiles of the tumor microenvironment in a T cell-dependent manner. In addition, CXCL14 expression is dramatically decreased in head and neck cancer, cervical, prostate, lung, pancreatic, gastric, and oral cancers.

[0050] However, CXCL14 cannot be directly used as a treatment agent due to its short half-life of about 30 minutes. To overcome this issue, the inventors have developed a fusion protein including CXCL14 and an immunoglobulin Fc region, which provides enhanced stability, possibly increasing the half-life to several weeks. This protein fusion has been successfully expressed in 293 cell culture and purified.

[0051] The Fc-CXCL14 fusion protein of the invention was shown to bind and increase proliferation of stellate cells, having activity superior to that of unconjugated CXCL14. Fig. 2 demonstrates that both CXCL14 (Fig. 2A) and Fc-CXCL14 (Fig. 2B) induce proliferation of LX-2 cells, however Fc-CXCL14 has a superior effect.

[0052] The Fc-CXCL14 fusion protein of the invention was further shown to bind and inhibit proliferation of HCC and breast cancer cells. Figs. 3-5 show that the Fc-CXCL14 is capable of inhibiting proliferation of cancer cell lines, including HCC cells FLC4 (Fig. 3A-3C) and Huh7 (Fig. 4A-4C), and breast cancer cell line MDA231 (Fig. 5A-5C) to a greater extent compared to CXCL14.

[0053] These results suggest that the Fc-CXCL14 fusion protein may be suitable as a cancer treatment agent.

[0054] The Fc-CXCL14 fusion protein

[0055] In some embodiments, the present invention provides a protein including a CXCL14 sequence linked to an immunoglobulin Fc region sequence.

[0056] The term “CXCL14 sequence” encompasses a sequence derived from a CXCL14 protein (e.g. human CXCL14 sequence of NCBI RefSeq ID No. NP_004878), having the same function or activity as a CXCL14 protein. The function or activity may be binding abilities of the CXCL14 protein, such as binding to stellate cells or to cancer cells, or abilities to increase stellate cells proliferation or to inhibit cancer cells proliferation.

[0057] The term “derived from”, as used herein, means that a first sequence derived from a second sequence has at least a certain level of sequence identity to the second sequence. In some embodiments, the certain level of sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0058] In some embodiments, the CXCL14 sequence is derived from a human sequence.

[0059] In some embodiments, the CXCL14 sequence includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence in the NCBI Reference Sequence ID No. NP_004878.

[0060] In some embodiments, the CXCL14 sequence includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 1.

[0061] In some embodiments, the CXCL14 sequence has a length of about 50-150, 50-120, 60-120, or 70-110 amino acids.

[0062] As explained above, chemokines are unstable and short lived. An immunoglobulin Fc region is large and heavy and confers increased stability. The addition of an immunoglobulin Fc region to the chemokine causes dimerization of the CXCL14 portion, making it more stable and therefore more active than the unfused CXCL14, thereby prolonging its half-life.

[0063] The term “immunoglobulin Fc region sequence” (herein Fc sequence) encompasses a sequence derived from a constant region of an immunoglobulin heavy chain. The Fc sequence may include the Fc hinge region and typically includes the second and third constant regions (CH2 and CH3) with or without the hinge region.

[0064] In some embodiments, the Fc sequence is derived from a human sequence. In some embodiments, the Fc sequence is derived from a non-human animal such as a cow, a goat, a swine, a mouse, a rabbit, a hamster, a rat and a guinea pigs.

[0065] In some embodiments, the Fc sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to an immunoglobulin Fc region sequence of an immunoglobulin selected from IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, and combinations thereof. In some embodiments, the immunoglobulin is IgG. In some embodiments, the immunoglobulin is IgGl. In some embodiments, the Fc sequence is derived from an Fc region of IgGl.

[0066] In some embodiments, the Fc sequence includes a complete immunoglobulin Fc region sequence. In some embodiments, the Fc sequence lacks the CHI region of the immunoglobulin Fc region, in order to prevent antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc sequence includes the hinge-CH2-CH3 regions of an immunoglobulin heavy chain and not the CHI region.

[0067] In some embodiments, the Fc sequence contains a modified glycosylation level compared to the native state in an antibody. For example, a lower glycosylation may result in a lower affinity to the complement system components, as well as a lower ability for ADCC. Reducing glycosylation levels may be done by methods known in the art including chemical methods, enzymatic methods, or by choice of the host for producing the fusion protein.

[0068] In some embodiments, the Fc sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence in UniProt accession No. P01857, P01857-1, or P01857-2.

[0069] The Fc sequence may include modifications compared to the P01857 (or P01857-1 or P01857-2) sequence, such as modifications which reduce binding to Fc receptors on cells, so as to abolish immune effector functions such as ADCC. Examples for such modifications include any of the following or combinations thereof: L234A, L235A, and P329G (amino acid positions are based on P01857-1). In some embodiments, the Fc sequence is from hlgGl-LALA-PG amino acids 104-330 (NCBI Protein database ID No. 6S5A_D).

[0070] In some embodiments, the immunoglobulin Fc sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 2.

[0071] In some embodiments, the immunoglobulin Fc sequence has a length of about 150-400, 200- 300m or 200-250 amino acids.

[0072] In some embodiments, the Fc sequence is N-terminal to the CXCL14 sequence in the fusion protein. In some embodiments, the Fc sequence is C-terminal to the CXCL14 sequence in the fusion protein. In order to allow flexibility of the fusion protein, the CXCL14 and the Fc sequence may be linked by a flexible linker, such as a peptide rich in glycine, or a combination of glycine with serine and / or with threonine. Generally, any suitable linker may be used, which allows flexibility of the protein and maintains the CXCL14 function.

[0073] Accordingly, in some embodiments, the fusion protein further includes a linker linking the CXCL14 sequence and the FC sequence. In some embodiments, the linker is rich in glycine, serine and / or threonine. In some embodiments, the linker has at least 70%, 75%, 80%, 85%, 90%, or 95% glycine, serine and / or threonine out of the total amino acids of the linker.

[0074] In some embodiments, the linker has a length of about 4-40, 4-35, 4-30, or 5-25 amino acids.

[0075] In some embodiments, the linker has a sequence at least 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 3.

[0076] The fusion protein may include additional sequences, such as targeting sequences, tags for detection, a secretion signal, etc.

[0077] In some embodiments, the fusion protein further includes a signal peptide at the N-terminus of the fusion protein, to direct the fusion protein for secretion.

[0078] In some embodiments, the signal peptide includes a PBG-SP1 sequence.

[0079] In some embodiments, the fusion protein includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 5.

[0080] It is appreciated that the fusion protein may further contain modifications including unconventional amino acids, modified amino acids, cyclization, capping, etc., as such modifications may confer additional protection and stability to the fusion protein.

[0081] When the fusion protein is used for treatment, as further disclosed below, the fusion protein may be conjugated to another active agent, for example, an anti-cancer drug, similar to an antibody-drug conjugate (ADC). The CXCL14 may help to target the anti-cancer drug to a tumor, where it will be most effective and less harmful to healthy cells. Many examples are known in the art for antibody-drug conjugates.

[0082] In some embodiments, the fusion protein is conjugated to an anti-cancer drug.

[0083] The anti-cancer drug may be a chemotherapeutic drug, an immunotherapeutic drug, or a radiotherapeutic drug.

[0084] In some embodiments, the fusion protein is capable of binding to stellate cells. In some embodiments, the fusion protein is capable of binding to cancer cells. In some embodiments, the fusion protein is capable of binding to hepatocellular carcinoma cells. In some embodiments, the fusion protein is capable of binding to breast cancer cells.

[0085] In some embodiments, the fusion protein is capable of increasing stellate cells proliferation. In some embodiments, the fusion protein is capable of inhibiting cancer cells proliferation. In some embodiments, the fusion protein is capable of inhibiting hepatocellular carcinoma cells proliferation. In some embodiments, the fusion protein is capable of inhibiting breast cancer cells proliferation.

[0086] In some embodiments, the fusion protein is capable of binding to a receptor selected from CCR2, CCR5, and CXCR4.

[0087] Nucleic acids, vectors, cells, and compositions including the fusion protein of the invention

[0088] In some embodiments, the present invention provides a nucleic acid molecule including a nucleotide sequence encoding the fusion protein disclosed herein.

[0089] Such nucleic acid molecules may further include elements needed for expression and / or regulation of expression of the fusion protein such as a transcription promoter, a transcription terminator, enhancers, etc. Such nucleic acid molecules may further include elements required for cloning of the fusion protein, such as suitable vector sequences and selection markers for cloning and expression in a desired cell.

[0090] In some embodiments, the nucleic acid molecule includes a sequence encoding the peptides of SEQ ID NOs: 1 and 2. In some embodiments, the nucleic acid molecule includes a sequence encoding the protein of SEQ ID NO: 5.

[0091] It is appreciated that the sequences encoding the peptides of SEQ ID NOs: 1 and 2 may be at any order. In some embodiments, the sequence encoding the peptide of SEQ ID NOs: 1 is N- terminal to the sequence encoding the peptide of SEQ ID NOs: 2. In some embodiments, the sequence encoding the peptide of SEQ ID NOs: 2 is N-terminal to the sequence encoding the peptide of SEQ ID NOs: 1.

[0092] In some embodiments, the nucleic acid molecule includes a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the nucleic acid molecule includes two sequences, one at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6 and another at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7.

[0093] It is appreciated that the sequences at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 6 and 7 may be at any order. In some embodiments, the sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 6 is N-terminal to the sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 7. In some embodiments, the sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 7 is N-terminal to the sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 6.

[0094] In some embodiments, the nucleic acid molecule further includes sequences encoding the peptides of SEQ ID NO: 3 and 4. In some embodiments, the nucleic acid molecule further includes sequences which are at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 8 and / or SEQ ID NOs: 9.

[0095] In some embodiments, the present invention provides a vector including the nucleic acid molecule disclosed herein. The vector may be any vector for delivery to mammalian cells including a viral vector, such as a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus (AAV); or a non-viral vector, such as a plasmid.

[0096] In some embodiments, the present invention provides a cell including the nucleic acid molecule disclosed herein, and / or expressing the fusion protein disclosed herein. The cell may be any cell suitable for expression or production of the fusion protein, such as, e.g., HEK-293T cells. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The cell may further be suitable for producing the fusion protein in a body, e.g., a human body. For example, the cell may be a human cell of a patient, when the cell is treated ex vivo by insertion of the nucleic acid into the cell to enable the cell to express and secrete the fusion protein in the body of the patient. The cell may be a blood cell, such as a white blood cell. The cell may be an immune system cell. Some nonlimiting examples for immune system cells suitable for the invention include T cells, B cells, NK cells, macrophages, and monocytes.

[0097] In some embodiments, the present invention provides a pharmaceutical composition including the fusion protein disclosed herein, the nucleic acid molecule of disclosed herein, the vector disclosed herein, or the cell disclosed herein, and a pharmaceutically acceptable carrier.

[0098] In some embodiments, the present invention provides the pharmaceutical composition disclosed herein, for use in a method of treating cancer in a subject in need thereof.

[0099] In some embodiments, there is provided the fusion protein disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0100] In some embodiments, there is provided the nucleic acid disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0101] In some embodiments, there is provided the vector disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0102] In some embodiments, there is provided the cell disclosed herein for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

[0103] Definitions and embodiments mentioned above and which may be relevant to the nucleic acid, vector, cell, composition, or use, embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0104] The pharmaceutical composition of the present invention may be formulated in any conventional manner using one or more physiologically or pharmaceutically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition, not being deleterious to the recipient thereof, and not significantly interfering with the activity of the polypeptide of the invention, or of any other active ingredient in the pharmaceutical composition. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0105] Method of producing the Fc-CXCL14 fusion protein

[0106] In some embodiments, the Fc-CXCL14 fusion protein is produced by transfecting suitable cells, e.g., HEK-293T cells, by a vector encoding the fusion protein and purifying it on a Protein A column. The transfection and purification may be carried out by any suitable method known in the art. The identity of the isolated protein may be confirmed by any method known in the art, such as ELISA or flow cytometry. For the identity confirmation, antibodies against CXCL14 or antibodies against immunoglobulin Fc may be used.

[0107] Definitions and embodiments mentioned above and which may be relevant to the methods of preparation embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0108] Method of treating cancer or metabolic syndrome diseases by administering the Fc-CXCL14 fusion protein

[0109] In some embodiments, the present invention provides a method of treating a cancer or a metabolic syndrome disease in a subject in need thereof, the method including administering to the subject the pharmaceutical composition disclosed herein.

[0110] In some embodiments, the present invention provides a method of treating a cancer or a metabolic syndrome disease in a subject in need thereof, the method including administering to the subject the fusion protein, nucleic acid, vector, and / or cell disclosed herein. Definitions and embodiments mentioned above and which may be relevant to the methods of treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0111] In some embodiments, the cancer is a solid tumor.

[0112] The cancer may be selected from, without being limited to, Adrenocortical carcinoma, Anal cancer, Appendix cancer, Astrocytoma (cerebellar, cerebral, childhood), Basal cell carcinoma, Bile duct cancer (extrahepatic), Bladder cancer, Bone cancer (Osteosarcoma, Malignant fibrous histiocytoma), Brainstem glioma, Brain tumor, Ependymoma, Medulloblastoma, Pineal tumors (astrocytoma, germinoma, pineoblastoma), Supratentorial primitive neuroectodermal tumors (PNET), Visual pathway and hypothalamic glioma (childhood), Glioma, Central nervous system lymphoma (primary), Bladder cancer, Renal cell carcinoma (kidney cancer), Transitional cell carcinoma (renal pelvis and ureter), Ureter cancer, Urethral cancer, Osteosarcoma, Malignant fibrous histiocytoma of bone, Ewing's sarcoma, Rhabdomyosarcoma (childhood), Desmoplastic small round cell tumor, Soft tissue sarcoma, Breast cancer, Esophageal cancer, Gastric (stomach) cancer, Gastrointestinal stromal tumor (GIST), Gastrointestinal carcinoid tumor, Colon cancer, Rectal cancer, Small intestine cancer, Liver cancer (hepatocellular carcinoma, HCC), Gallbladder cancer, Pancreatic cancer, Islet cell carcinoma (endocrine pancreas), Parathyroid cancer, Thyroid cancer, Multiple endocrine neoplasia syndrome (childhood), Intraocular melanoma, Retinoblastoma, Cervical cancer, Endometrial (uterine) cancer, Ovarian cancer (epithelial, germ cell, low malignant potential), Vaginal cancer, Vulvar cancer, Uterine sarcoma, Gestational trophoblastic tumor, Prostate cancer, Penile cancer, Testicular cancer, Head and neck cancer, Laryngeal cancer, Lip and oral cavity cancer, Mouth (oral) cancer, Oropharyngeal cancer, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Pharyngeal cancer, Throat cancer, Salivary gland cancer, Heart cancer, Hodgkin lymphoma, Non-Hodgkin lymphoma, Burkitt lymphoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorders, Hairy cell leukemia, Mycosis fungoides, Sezary syndrome, Waldenstrom macroglobulinemia, Wilms tumor (childhood kidney cancer), Non-small cell lung cancer, Small cell lung cancer, Bronchial adenomas / carcinoids, Pleuropulmonary blastoma, Mesothelioma (adult and childhood), Neuroblastoma, Pheochromocytoma, Carcinoma of unknown primary, Metastatic squamous neck cancer with occult primary, Childhood cancers, Unknown primary site carcinoma (adult and childhood), Skin cancer (nonmelanoma, melanoma), Merkel cell carcinoma, Cutaneous T-cell lymphoma, Kaposi sarcoma, and uterine sarcoma,.

[0113] In some embodiments, the cancer is selected from HCC, breast cancer (e.g. triple negative breast cancer), head and neck cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, and renal cancer.

[0114] In some embodiments, the metabolic syndrome diseases is selected from obesity, insulin resistance, type 2 diabetes, hyperglycemia, dyslipidemia, elevated triglycerides, low HDL cholesterol, hypertension, non-alcoholic fatty liver disease (NAFLD), atherosclerosis, cardiovascular disease, chronic kidney disease (CKD), sleep apnea, gout, polycystic ovary syndrome and proinflammatory state.

[0115] In some embodiments, the method further includes treating with an anti-cancer treatment. The anti-cancer treatment may include any anti-cancer treatment or anti-cancer agent or drug, including radiotherapy, chemical therapy agents, biological agents, immunological agents (e.g. monoclonal antibodies), etc.

[0116] When treating with an anti-cancer treatment, the pharmaceutical composition disclosed herein and the anti-cancer treatment may be administered at any arrangement with respect to one another. For example, the anti-cancer treatment may be administered prior to administration of the Fc-CXCL14, following administration of the CXCL14, at the same time as administration of the Fc-CXCL14 (simultaneous - when the agent is part of the same composition or is part of a separate compositions), or both the Fc-CXCL14 and the anti-cancer treatment may be administered as part of the same administration regiment. In some embodiments, the anti-cancer agent is conjugated to the CXCL14.

[0117] In some embodiments, the anti-cancer agent is administered at a sub-therapeutic dose. The term “sub-therapeutic dose”, as used herein, relates to a dose that is not expected to be effective for treating the same condition when the anti-cancer agent is administered without the Fc-CXCL14 of the invention.

[0118] In some embodiments, the effect of treatment with both the Fc-CXCL14 and the anti-cancer treatment is more effective than treating with the anti-cancer treatment alone, leading, e.g. to a faster or a greater reduction in tumor size, or an increased immune response against the tumor.

[0119] The term “treating” or “treatment”, as used herein, refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a cancer and / or symptoms attributed to the cancer. The term includes inhibiting the tumor growth or spread (invasion / metastasis), i.e. arresting its development; or ameliorating the cancer, i.e. causing regression of the cancer, e.g., by eliminating or ameliorating its symptoms.

[0120] The administration may be a systemic or a local administration. Non-limiting examples for administration routes include intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, oral, sublingual, enteral, intranasal, buccal, vaginal, rectal, intraocular, intrathecal, topical, transdermal, intratumoral, and intradermal administration.

[0121] In some embodiments, the route of administration is selected from intravenous, subcutaneous, intramuscular, intratumoral, intraperitoneal, intranasal, and topical administration.

[0122] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0123] The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0124] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0125] The terms “protein”, “peptide”, and “polypeptide” as used herein are intended to encompass amino acid sequences that are naturally occurring as well as those recombinantly or synthetically produced. The proteins, peptides, and polypeptides (hereinbelow “entities”) of the invention also encompass those including conservative amino acid substitutions with respect to claimed entities as long as they have the same activity as the unmodified entities. Given the known properties of individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., “conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used.

[0126] Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0127] The following six groups each contain amino acids that are conservative substitutions for one another: 1 - Alanine (A), Serine (S), Threonine (T); 2- Aspartic acid (D), Glutamic acid (E); 3- Asparagine (N), Glutamine (Q); 4- Arginine (R), Lysine (K); 5- Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6- Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0128] The entities of the present invention may be produced by any method known in the art, including recombinant and synthetic methods. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. Solid phase peptide synthesis procedures are well known to one skilled in the art and described, for example by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic peptides are purified by preparative high-performance liquid chromatography (Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.) The peptide sequence may be confirmed by amino acid sequencing using methods known to one skilled in the art.

[0129] In some embodiments, recombinant protein techniques are used to generate the entities of the present invention. Recombinant techniques are described for example by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0130] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0131] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

[0132] Table 1: Protein sequences

[0133] The highlighted amino acids in SEQ ID NO: 2 are the hlgGl-LALA-PG substitutions L234A,

[0134] L235A, and P329G with respect to UniProt accession No. P01857-1.

[0135] Table 2: Nucleic acid sequences

[0136] Example 1: Preparation and purification of the fusion protein Fc-CXCL14

[0137] A vector encoding the Fc-CXCL14 fusion protein of SEQ ID NO: 5 was prepared by preparing and cloning a sequence encoding the following amino acid sequences (from the N- terminus): PBG-SP1 signal sequence from kappa immunoglobulin light chain (SEQ ID NO: 4); methionine; human IgGl Fc hinge-CH2-CH3 regions positions 104-330 of P01857-1 including the substitutions L234A, L235A, and P329G (SEQ ID NO: 2); a GS-linker (SEQ ID NO: 3); and amino acids positions 23-99 of the human CXCL14 protein in NCBI Reference Sequence: NP_004878 (SEQ ID NO: 1), into an Xbal / EcoRV cloning site of a pcDNA™3.4 plasmid vector under CMV promoter. The vector was transformed into a culture of Expi293™ cells, and cells were grown for 5 days in 36.5°C in orbital shaking. Media were collected from the cells, and the Fc-CXCL14 fusion-protein was cleaned from the media on a Protein A column (Fig. 1A). Fig. IB shows that the Fc-CXCL14 fusion protein could be detected by the same antibodies used for detection of the CXCL14 protein by ELISA. CXCL12 was used as a negative control. Example 2: Fc-CXCL14 increases proliferation of the stellate LX-2 cell line.

[0138] 104LX-2 cells / well were incubated with increasing doses of CXCL14 (Fig. 2A) or Fc- CXCL14 (Fig. 2B): Ing / ml, lOng / ml, 100 ng / ml, or lOOOng / ml. Plates were photographed every 3 hours by the IncuCyte® Live-Cell Analysis System and proliferation was analyzed by the IncuCyte® software. When comparing Fig. 2A to Fig. 2B, it can be seen that the effect of the Fc- CXCL14 fusion protein on cell proliferation is stronger than that of the CXCL14 protein.

[0139] Example 3: Fc-CXCL14 binds to HCC FLC4 cells and decreases proliferation.

[0140] 106FLC4 cells (a human hepatocellular carcinoma (HCC) cell line) were incubated with Fc- CXCL14 at room temperature (RT) for 1 hour. Cells were then incubated with an anti-FC antibody conjugated to Phycoerythrin (PE) at 4°C in the dark for 20 min. Mean fluorescence intensity (MFI) levels were measured by flow cytometry (Fig. 3A).

[0141] 104FLC4 cells / well were incubated with different doses of CXCL14 (Fig. 3B) or Fc- CXCL14 (Fig. 3C): Ing / ml, lOng / ml, 100 ng / ml, and lOOOng / ml. Plates were photographed every 3 hours by the IncuCyte® Live-Cell Analysis System and proliferation was analyzed by the IncuCyte® software. Decreased proliferation with the addition of CXCL14 can be seen, with a stronger effect for the Fc-CXCL14 fusion protein.

[0142] Table 3: % proliferation inhibition:

[0143] Example 4: Fc-CXCL14 binds to HCC huh7 cells and decreases proliferation.

[0144] 106Huh7 cells were incubated with Fc-CXCL14 at room temperature (RT) for 1 hour. Cells were then incubated with an anti-FC antibody conjugated to Phycoerythrin (PE) at 4°C in the dark for 20 min. Mean fluorescence intensity (MFI) levels were measured by flow cytometry (Fig. 4A).

[0145] 104of Huh7 cells / well were incubated with different doses of CXCL14 (Fig. 4B) or Fc- CXCL14 (Fig. 4C): Ing / ml, lOng / ml, 100 ng / ml, and lOOOng / ml. Plates were photographed every 3 hours by the IncuCyte® Live-Cell Analysis System and proliferation was analyzed by the IncuCyte® software. Decreased proliferation with the addition of CXCL14 can be seen, with a stronger effect for the Fc-CXCL14. Table 4: % proliferation inhibition:

[0146] Example 5: Fc-CXCL14 decreases proliferation in breast and colon cancer cells.

[0147] 106MDA-231 breast cancer cells were incubated with Fc-CXCL14 at room temperature (RT) for 1 hour. Cells were then incubated with an anti-FC antibody conjugated to Phycoerythrin (PE) at 4°C in the dark for 20 min. Mean fluorescence intensity (MFI) levels were measured by flow cytometry (Fig. 5A (MDA-231)).

[0148] 104of MDA-231 or MDA468 breast cancer cells / well were incubated with different doses of CXCL14 (Figs. 5B (MDA-231) and 6A (MDA468)) or Fc-CXCL14 (Figs. 5C (MDA-231) and 6B (MDA468)): Ing / ml, lOng / ml, 100 ng / ml, and lOOOng / ml. Plates were photographed every 3 hours by the IncuCyte® Live-Cell Analysis System and proliferation was analyzed by the IncuCyte® software. Decreased proliferation with the addition of CXCL14 can be seen, with a stronger effect for the Fc-CXCL14.

[0149] Table 5: % proliferation inhibition for MDA-231 cells:

[0150] A similar experiment was conducted with DLD1 colon cancer cells. The results are presented in Figs. 7A-7B, showing decreased proliferation of the cells in a dose dependent manner for cells treated with Fc-CXCL14 but not for cells treated with CXCL14.

[0151] Example 6: CXCL14 binding to LX-2 Stellate Cells is independent of CXCR4.

[0152] 106LX-2 cells were incubated with Fc-CXCL14 at RT for 1 hour, or with SDF-1 (CXCL12), the CXCR4 ligand used here to block CXCR4 at RT for 1 hour, and then with Fc-CXCL14. Cells were then incubated with anti-FC antibody conjugated to PE at 4°C in the dark for 20 min. MFI levels were measured by Flow cytometry. Fig. 8 shows that pre-binding with SDF-1 does not inhibit Fc-CXCL14 binding, indicating that Fc-CXCL14 does not bind to stellate cells through the CXCR4 receptor.

[0153] Example 7: Ligands for chemokine receptors CCR2 and CCR5 inhibit the binding of Fc- CXCL14 to the stellate LX-2 cell line.

[0154] 106LX-2 cells / well were incubated with the CCR2, CCR5, and CXCR4 chemokine ligands:- correct SDF-1 (CXCL12), RANTES, MCP-1, MIPla, MIPlb, MIP3b, SLC and SCF, and combinations thereof, at RT for 1 hour, followed by incubating with Fc-CXCL14 at RT for 1 hour. Cells were then incubated with an anti-FC antibody conjugated to PE at 4°C in the dark for 20 min. MFI levels were measured by Flow cytometry. As can be seen from Fig. 9, some of the chemokines, especially the combination of MCP-1 (a CCR2 ligand) and MIPla (a CCR5 ligand), inhibited Fc-CXCL14 binding, indicating that CXCL14 may bind to stellate cells through the CCR2 and / or CCR5 receptors.

[0155] Example 8: Ligands for the chemokine receptors CCR2, CCR5 and CXCR4 inhibit the binding of Fc-CXCL14 to THP-1 cell line.

[0156] 106THP-1 (a childhood acute monocytic leukemia -derived cell line which is also a model for monocytes) cells / well were incubated with the CCR2, CCR5, and CXCR4 chemokine ligands and others: SDF-1 (CXCL12), RANTES, MCP-1, MIPla, MIPlb, MIP3b, SLC and SCF, and combinations thereof, at RT for 1 hour, followed by incubating with Fc-CXCL14 at RT for 1 hour. Cells were then incubated with an anti-FC antibody conjugated to PE at 4°C in the dark for 20 min. MFI levels were measured by Flow cytometry. As can be seen from Fig. 10, some of the chemokines, especially SDF-1 (a CXCL4 ligand), RANTES (a CCR5 ligand), and combinations of MCP1 (a CCR2 ligand), MIPla (a CCR5 ligand), and MIPlb (a CCR5 ligand), inhibited Fc- CXCL14 binding, indicating that CXCL14 may bind to monocytes through the CXCR4, CCR2 and / or CCR5 receptors.

[0157] Example 9: Safety testing the Fc-CXCL14 fusion

[0158] The safety of Fc-CXCL14 was tested in NSG (NOD SCID gamma) mice. lOOug of Fc- CXCL14 were administered to the mice by an i.v. injection, and blood levels of various blood components were tested 4 hours, 24 hours, 3 days, 6 days and 10 days following the injection, compared to a control (mice injected with PBS for measuring background, confirming the specificity of the ELISA). Fig. 11A confirms the presence of the fusion protein and shows that The levels of the fusion protein remain high compared to the control group. Fig. 11B shows no significant changes in weight of organs (spleen, liver, lung, heart, and kidney) between the two groups. Fig. 11C shows blood test results 13 days post injection, generally exhibiting no significant changes between controls and mice injected with Fc-CXCL14 in parameters including white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.

[0159] Example 10: Testing the Fc-CXCL14 in vivo in an HCC mouse xenograft model

[0160] Tumor cells of hepatocellular carcinoma (HCC) cell line such as FLC4, HEPG2, or Huh7, or tumor cells from an HCC cancer of a patient, are subcutaneously injected into immune deficient mice such as nude mice or SCID mice.

[0161] The rate of growth is measured by an IVIS camera and a caliper. Various amounts of the Fc- CXCL14 fusion protein of the invention are injected intraperitoneally into the mice, and the relationship between the amount of fusion protein injected and tumor dimensions such as length, volume, and weight, are examined. Further assessments include histochemistry, and localization of the fusion protein.

[0162] It is expected that tumor dimensions will be reduced following administration of the Fc- CXCL14 fusion protein, indicating suitability for treatment of cancer.

[0163] Example 11: Testing the Fc-CXCL14 in vivo in a breast cancer mouse xenograft model

[0164] Tumor cells from a breast cancer cell line such as MDA231, or tumor cells from an breast cancer of a patient, are subcutaneously injected into immune deficient mice such as nude mice or SCID mice.

[0165] The rate of growth is measured by an IVIS camera and a caliper. Various amounts of the Fc- CXCL14 fusion protein of the invention are injected intraperitoneally into the mice, and the relationship between the amount of fusion protein injected and tumor dimensions such as length, volume, and weight, are examined. Further assessments include histochemistry, and localization of the fusion protein.

[0166] It is expected that tumor dimensions will be reduced following administration of the Fc- CXCL14 fusion protein, indicating suitability for treatment of cancer.

[0167] Example 12: Testing the effect of Fc-CXCL14 on immune response against tumors

[0168] A culture of tumor cells combined with peripheral blood lymphocytes (PBMC)s is prepared, in which the PBMCs are activated against the tumor cells. The tumor cells may be obtained from a cancer patient or from a tumor cell line as mentioned above. The PBMCs may further be activated by treating with anti-CD3 and / or IL2.

[0169] Various amounts of the Fc-CXCL14 fusion protein are added to the system and the effect of the fusion protein on various factors of the immune response against the tumor is tested by measuring cancer cells death compared to cells with PBMCs only. The experiments may be done by further adding an immunotherapy, such as an immune checkpoint inhibitor (such as Pembrolizumab (e.g. Keytruda)).

[0170] Example 13: Testing the effect of Fc-CXCL14 on lipid metabolism and liver damage

[0171] Mice are fed with a high fat (HFD) diet. After 7 weeks, mice are injected intraperitoneally with various amounts of the Fc-CXCL14 fusion protein, and the relationships between the amount injected and parameters of lipid metabolism and / or of liver damage including weight, blood glucose levels, and white fat vs. brown fat levels, are tested.

[0172] It is expected that the lipid metabolism and / or liver damage parameters will be reduced following administration of the Fc-CXCL14 fusion protein, indicating suitability for treatment of metabolic syndrome disease.

Claims

CLAIMSWhat is claimed is:

1. A fusion protein comprising a CXCL14 sequence linked to an immunoglobulin Fc region sequence.

2. The fusion protein of claim 1, wherein at least one of the CXCL14 sequence and the Fc region sequence is derived from a human sequence.

3. The fusion protein of claim 1 or 2, wherein the CXCL14 sequence comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1.

4. The fusion protein of any one of claims 1-3, wherein the CXCL14 sequence has a length of about 50-150, 50-120, 60-120, or 70-110 amino acids.

5. The fusion protein of any one of claims 1-4, wherein the immunoglobulin Fc region sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to an immunoglobulin Fc region of an immunoglobulin selected from IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.

6. The fusion protein of any one of claims 1-5, wherein the immunoglobulin Fc region sequence comprises a complete immunoglobulin Fc region sequence.

7. The fusion protein of any one of claims 1-5, wherein the immunoglobulin Fc region sequence lacks an CHI region of the immunoglobulin Fc region, in order to prevent antibody-dependent cellular cytotoxicity (ADCC).

8. The fusion protein of any one of claims 1-7, wherein the immunoglobulin Fc region sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 2.

9. The fusion protein of any one of claims 1-8, wherein the immunoglobulin Fc region sequence is N-terminal to the CXCL14 sequence in the fusion protein.

10. The fusion protein of any one of claims 1-9, further comprising a linker between the immunoglobulin Fc region sequence and the CXCL14 sequence.

11. The fusion protein of claim 10, wherein the linker is a flexible linker rich in glycine, serine and / or threonine.

12. The fusion protein of claim 10 or 11, wherein the linker has a length of about 4-40, 4-35, 4-30, or 5-25 amino acids.

13. The fusion protein of any one of claims 10-12, wherein the linker has a sequence at least 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 3.

14. The fusion protein of any one of claims 1-13, further comprising a signal peptide at the N- terminus of the fusion protein.

15. The fusion protein of any one of claims 1-14, comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence as set forth in SEQ ID NO: 5.

16. The fusion protein of any one of claims 1-15, wherein the fusion protein is conjugated to an anti-cancer drug.

17. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-15.

18. A vector comprising the nucleic acid molecule of claim 17.

19. A cell comprising the nucleic acid molecule of claim 17 or the vector of claim 18, and / or expressing the fusion protein of any one of claims 1-15.

20. A pharmaceutical composition comprising the fusion protein of any one of claims 1-16, the nucleic acid molecule of claim 17, the vector of claim 18, and / or the cell of claim 19, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

22. The fusion protein of any one of claims 1-16, for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

23. The nucleic acid of claim 17, for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

24. The vector of claim 18, for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

25. The cell of claim 19, for use in a method of treating cancer or a metabolic syndrome disease in a subject in need thereof.

26. A method of treating a cancer or a metabolic syndrome disease in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 20.

27. The pharmaceutical composition for use of claim 21 or the method of claim 26, wherein the cancer is selected from hepatocellular carcinoma (HCC), breast cancer, head and neck cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, and renal cancer.

28. The pharmaceutical composition for use of claim 21 or the method of claim 26, wherein the metabolic syndrome disease is selected from obesity, insulin resistance, type 2 diabetes, hyperglycemia, dyslipidemia, elevated triglycerides, low HDL cholesterol, hypertension, nonalcoholic fatty liver disease (NAFLD), atherosclerosis, polycystic ovary syndrome and sleep apnea.

29. The pharmaceutical composition for use of claim 21 or the method of claim 26, wherein the treating comprises administering the pharmaceutical composition of claim 20 in combination with an anti-cancer treatment.

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