阅读说明：本技术 靶向细胞中c-Myc的嵌合分子 (Chimeric molecules targeting c-Myc in cells ) 是由 法德利克·巴德 A·肖梅特 陈媛 于 2018-11-29 设计创作，主要内容包括：本文公开了嵌合融合蛋白,其包含与遗传修饰的铜绿假单胞菌(Pseudomonas Aeroginosa)外毒素A(‘tPE’)融合的c-Myc抑制剂,所述融合蛋白能够穿透细胞的细胞核并抑制细胞核内的c-Myc活性。具体地,tPE包含PE结构域Ia和II,且c-Myc抑制剂是衍生自c-Myc的螺旋1羧基区域的H1肽。本文还公开了包含嵌合融合蛋白的药物组合物,嵌合融合蛋白的递送方法、制备方法以及治疗c-Myc依赖性癌症的用途。(Disclosed herein are chimeric fusion proteins comprising a c-Myc inhibitor fused to a genetically modified Pseudomonas aeruginosa (Pseudomonas Aeroginosa) exotoxin a ('tPE'), which fusion protein is capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus. Specifically, tPE comprises PE domains Ia and II, and the c-Myc inhibitor is an H1 peptide derived from the helix 1 carboxy region of c-Myc. Also disclosed herein are pharmaceutical compositions comprising the chimeric fusion proteins, methods of delivery, methods of preparation, and uses for treating c-Myc dependent cancers of the chimeric fusion proteins.)

1. A chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified Pseudomonas aeruginosa (Pseudomonas Aeroginosa) exotoxin a ('tPE'), said fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within said nucleus.

2. A pharmaceutical composition comprising a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), said fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within said nucleus.

3. A method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the step of subjecting a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to the cell, wherein the fusion protein is capable of penetrating the nucleus of the cell and inhibiting c-Myc activity within the nucleus.

4. A method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the steps of: fusing the c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of the cell and inhibiting c-Myc activity within the nucleus, and subjecting the fusion protein to the cell.

5. A method of making a chimeric fusion protein capable of penetrating the nucleus of a cell, the method comprising the step of fusing a c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of the cell and inhibiting c-Myc activity within the nucleus.

6. A method of preventing or treating a c-Myc-dependent cancer in a subject, the method comprising the step of administering to the subject a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

7. A chimeric fusion protein for preventing or treating a c-Myc-dependent cancer in a subject, wherein the chimeric fusion protein comprises a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

8. Use of a chimeric fusion protein in the manufacture of a medicament for preventing or treating a c-Myc-dependent cancer in a subject, wherein the chimeric fusion protein comprises a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

9. The fusion protein of claim 1 or 7, the pharmaceutical composition of claim 2, the method of any one of claims 3-6, or the use of claim 8, wherein the c-Myc inhibitor: is capable of disrupting c-Myc-dependent pathways in c-Myc-dependent cancers; interference with specific c-Myc DNA binding; alternatively, c-Myc/Max dimerization is blocked.

10. The fusion protein of claim 1, 7 or 9, the pharmaceutical composition of claim 2 or 9, the method of any one of claims 3 to 6 and 9, or the use of claim 8 or 9, wherein the C-Myc inhibitor is fused to the C-terminus of tPE.

11. The fusion protein according to claim 1, 7, 9 or 10, the pharmaceutical composition according to claim 2, 9 or 10, the method according to any one of claims 3 to 6 and 9 or 10, or the use according to any one of claims 8, 9 and 10, wherein said tPE comprises PE domain Ia or a biologically active fragment thereof, preferably having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and (c) the sequence shown in fig. 11.

12. The fusion protein according to claim 1, 7, 9 or 10, the pharmaceutical composition according to claim 2, 9 or 10, the method according to any one of claims 3 to 6 and 9 or 10, or the use according to any one of claims 8, 9 and 10, wherein said tPE comprises PE domain II or a biologically active fragment thereof, preferably having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or a sequence shown in figure 12.

13. The fusion protein according to claim 1, 7, 9 or 10, the pharmaceutical composition according to claim 2, 9 or 10, the method according to any one of claims 3 to 6 and 9 or 10, or the use according to any one of claims 8, 9 and 10, wherein said tPE comprises PE domain Ia and PE domain II, preferably having the amino acid sequence of SEQ ID NO: 10, or a fragment thereof.

14. The fusion protein of claim 13, the pharmaceutical composition of claim 13, the method of claim 13, or the use of claim 13, wherein the C-Myc inhibitor is fused to the C-terminus of domain II of tPE.

15. The fusion protein of claims 1, 7 or 9 to 14, the pharmaceutical composition of claims 2 or 9 to 14, the method of any one of claims 3 to 6 and 9 to 14, or the use of any one of claims 8 to 14, wherein the c-Myc inhibitor is an H1 peptide derived from the helix 1(H1) carboxy region of c-Myc.

16. The fusion protein of claim 15, the pharmaceutical composition of claim 15, the method of claim 15, or the use of claim 15, wherein the H1 peptide has the sequence NELKRAFAALRDQI (SEQ ID No. 5).

17. The fusion protein of any one of claims 1, 7, and 9 to 16, the pharmaceutical composition of any one of claims 2 and 9 to 16, the method of any one of claims 3 to 6 and 9 to 16, or the use of any one of claims 8 to 16, wherein the chimeric protein comprises an N-terminal polyhistidine tag.

18. The fusion protein of claim 1 or claim 7, the pharmaceutical composition of claim 2, the method of any one of claims 3 to 6, or the use of claim 8, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence shown in figure 15.

19. The fusion protein of any one of claims 1, 7 and 9 to 18, the pharmaceutical composition of any one of claims 2 and 9 to 18, the method of any one of claims 3 to 6 and 9 to 18, or the use of any one of claims 8 to 18, wherein the cell is a cancer cell of the cervix, colon, breast, lung or stomach.

20. The method of claim 6, the fusion protein of claim 7, or the use of claim 8, wherein the subject is a human.

Technical Field

The present invention relates to the field of cancer, and more particularly, to compositions and methods for treating cancer using chimeric fusion proteins comprising genetically modified pseudomonas aeruginosa (pseudomonas aeroginosa) exotoxin a fused to a c-Myc inhibitor that can target and penetrate the nucleus and inhibit the activity of the nuclear transcription factor c-Myc.

Background

c-Myc is a regulatory gene encoding the transcription factor c-Myc. Mutant forms of c-Myc are found in many cancers, which result in constitutive expression of c-Myc and display oncogenic activity. This leads to deregulated expression of many genes, some of which are involved in cell proliferation and lead to the formation of cancer.

The frequency of c-myc gene alterations in human cancer has led to an estimated correlation of approximately 70,000 cancer deaths per year in the united states with alterations in the c-myc gene or its expression. Given that c-Myc may cause one-seventh of cancer deaths in the united states, recent efforts have been directed to inhibiting c-Myc protein in cancer biology, with the hope of therapeutic insight.

Thus, there is an unmet need for methods of detecting and/or targeting c-Myc in cells.

Disclosure of Invention

In one aspect, the invention relates to a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus.

In another aspect, the invention relates to a pharmaceutical composition comprising a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), said fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus.

In yet another aspect, the invention relates to a method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the step of subjecting to the cell a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), wherein the fusion protein is capable of penetrating the nucleus of the cell and inhibiting c-Myc activity within the nucleus.

In another aspect, the invention relates to a method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the steps of: fusing the c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus, and subjecting the fusion protein to the cell.

In another aspect, the invention relates to a method of making a chimeric fusion protein capable of penetrating the nucleus of a cell, the method comprising the step of fusing a c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus.

In another aspect, the invention relates to a method of preventing or treating a c-Myc-dependent cancer in a subject, the method comprising the step of administering to the subject a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

In one aspect, the invention relates to a chimeric fusion protein for preventing or treating a c-Myc-dependent cancer in a subject, wherein the chimeric fusion protein comprises a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

In another aspect, the invention relates to the use of a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), in the manufacture of a medicament for preventing or treating a c-Myc dependent cancer in a subject, wherein the fusion protein is capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

Drawings

Various embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1: tPE-Omomyc and tPE-H1 schematic of the primary structure and principles of the present invention. (A) The primary structures of tPE-Omomyc and tPE-H1, respectively, are shown. For the tPE-Omomyc fusion protein, PE domains Ia and II were genetically fused to an Omomyc peptide. For the tPE-H1 fusion protein, PE domains Ia and II were genetically fused to the H1 peptide. (B) Schematic diagrams of E-BOX under normal conditions and under c-Myc conditions are shown. When cells were incubated with tPE-Omomyc, the c-Myc/Max complex could no longer bind to the E-Box promoter. In a similar manner, neither the max peptide nor the c-Myc protein (bound to the tPE-H1 fusion protein) could bind to the E-Box promoter when the cells were incubated with tPE-H1. Cells expressed luciferase (luciferase) via the E-Box promoter to measure c-myc activity and Renilla luciferase (Renilla) via the CMV promoter as a control.

FIG. 2: subcellular localization. (A) A431 cell fractions after 1 hour tPE-H1200 nM treatment were shown to show the location of tPE-H1, c-Myc, and Max in the nucleus of the cell. C: a cytosolic fraction; m: a cell membrane moiety; n: a nuclear fraction. Antibodies were labeled on the left side of each blot. Molecular Weights (MW) are shown on the right. (B) A431 cell fractionation after 1 hour tPE-Omomyc 200nM treatment was shown to show tPE-Omomyc in the nuclear fraction. Alpha-tubulin is used as a cytosolic fraction marker, calnexin is used as a cell membrane fraction marker, and Max is used as a nuclear fraction marker. C: cytosolic fraction, M: cell membrane fraction, N: a nuclear fraction. Antibodies were labeled on the left side of each blot. MW is shown on the right. The results showed the presence of tPE-Omomyc in the nuclear fraction after 1 hour of incubation.

FIG. 3: tPE-H1 on the c-Myc/Max complex. In the absence of c-Myc antibody (-Ab, negative control) or in the presence of c-Myc antibody (IP c-Myc), co-immunoprecipitation of c-Myc was performed with Max. Cells were treated with tPE (i.e., PE domains Ia and II, without H1 peptide) and tPE-H1 to test for the effect of their c-Myc/Max interaction. The simulated sample did not contain any of the tPE forms. Antibodies were labeled on the left side of each blot. Molecular Weights (MW) are shown on the right.

FIG. 4: tPE-H1 dose response and EC for c-Myc transcriptional activity₅₀. At 6 hours, tPE-H1 responded to a dose of A431 cells expressing luciferase under the control of the E-Box promoter. Cells were treated with several concentrations of tPE-H1. Calculated EC₅₀＝25nM。

FIG. 5: tPE-H1 kinetics for c-Myc transcriptional activity. tPE-H150 nM on the kinetics of A431 cells expressing luciferase under the control of the E-Box promoter. Cells were treated and luciferase activity was read at various time points. The peak activity occurred at 6-8 hours and remained stable.

FIG. 6: tPE-H1 summarizes the results and the c-Myc specificity. A431 cells expressing luciferase under the control of the E-Box promoter and Renilla luciferase under the control of the CMV promoter were treated with 50nM tPE (negative control; SEQ ID NO: 10), tPE-H1(SEQ ID NO: 14) containing a mutant form of the H1 negative control, or tPE-H1 control (SEQ ID NO: 13) for 6 hours. The results show that luciferase is reduced when cells are treated with tPE-H1 instead of tPE or tPE-H1-control (black bars). Renilla luciferase was not affected by treatment (grey bars). The results show the specificity of tPE-H1 for E-Box luciferase.

FIG. 7: CPP-H1 dose response and EC for c-Myc transcriptional activity₅₀. Comparative dose responses after 6 hour treatment of cell targeting peptides (CPP) fused to H1 and tPE-H1 on A431 cells expressing luciferase under the control of the E-Box promoter. The X axis is shown at Log calnexin (CAD; LLIILLRRRIRKQAHAHSK; SEQ ID NO: 2) EC ₅₀75 μ M drosophila melanogaster podophide (antelopia) (Int; RQIKIWFQNRRMKWKK SEQ ID NO: 3) EC ₅₀200 μ M and TAT (GRKKRRQRRPPQ; SEQ ID NO:4) EC₅₀＝500μM。

FIG. 8: tPE-H1 on A431 cell proliferation. A431 cells were treated with 50, 100, 200 and 400nM tPE-H1 for more than 2 weeks. Brightfield collection and analysis were performed every 4 hours. The results showed that the cells proliferated slowly at 50nM and 100nM tPE-H1, and not above 200 nM.

FIG. 9: tPE-H1 on the proliferation of HepG2 cells from liver cancer. HepG2 cells were treated with 10nM, 25nM, 50nM and 100nMTPE-H1 for more than 2 weeks. Brightfield collection and analysis were performed every 4 hours. The results showed that the cells proliferated slowly at 10nM and 25nM tPE-H1, and no proliferation above 50 nM.

FIG. 10: mortality of liver cancer HepG2 treated with tPE-H1. HepG2 was treated with (black bars) or without (grey bars) 100nM tPE-H1 in the presence of the cell death marker DRAQ7 for 24 hours. Positive DRAQ7 cells were counted and compared under each condition. The results show an increase in the number of dead cells 24 hours after treatment with tPE-H1100 nM.

FIG. 11: tPE-H1 on c-Myc biomarkers. A431 cells were treated overnight with tPE-H1100 nM prior to RNA extraction. The amount of mRNA of transcripts regulated by c-Myc was quantified by RT-PCRQ, and compared with treatment with or without tPE-H1. Housekeeping mrnas (HPRT1, GAPDH) whose expression is not regulated by c-Myc were analyzed in the same way. The Y-axis shows mRNA log2 (fold change). Compared to housekeeping genes, the up-regulated genes showed negative log2 (fold change). Compared to housekeeping genes, down-regulated genes show positive log2 (fold change).

FIG. 12: tPE-Omomyc dose response and EC for transcriptional activity of c-Myc₅₀. Dose response of tPE-Omomyc to A431 cells expressing luciferase under the control of the E-BOX promoter at 6 hours (depicted as a line graph). Cells were treated with several concentrations of tPE-Omomyc, as shown on the X-axis of FIG. 12. The results show the calculated EC after 6 hours incubation₅₀This is 5nM lower than the results obtained with tPE-H1.

FIG. 13: tPE-Omomyc kinetics for c-Myc transcriptional activity. FIG. 13 shows a line graph depicting the kinetic effect of tPE-Omomyc 10nM on A431 cells expressing luciferase under control of the E-BOX promoter. Cells were treated and luciferase activity was read at various time points. The results show that the peak activity occurred 16 hours after incubation and remained stable.

FIG. 14: tPE-Omomyc effect on proliferation of liver cancer HepG2 cells. HepG2 cells were treated with 1nM, 2.5nM, 5nM and 10nM tPE-Omomyc for 2 weeks. Brightfield collection and analysis were performed every 4 hours. The results showed that the cells proliferated slowly at 2.5nM and 5 nMTBE-Omomyc, with no proliferation above 10 nM.

FIG. 15: liver cancer HepG2 cells treated with tPE-Omomyc died. HepG2 was treated with (black bars) or without (white bars) 10nM tPE-Omomyc for 24 hours in the presence of the cell death marker DRAQ 7. Positive DRAQ7 cells were counted and compared for each condition. The results show an increase in the number of dead cells 24 hours after treatment with tPE-Omomyc at a concentration of 10 nM.

Sequence Listing

Detailed Description

The present inventors have developed genetically modified forms of Pseudomonas aeruginosa (Pseudomonas Aeroginosa) exotoxin a ('tPE') that are capable of targeting and penetrating the nucleus of a cell and that can be used to target/deliver therapeutically/biologically active peptides or proteins to the nucleus. More specifically, the inventors have developed a chimeric fusion protein comprising a c-Myc inhibitor fused to tPE, which tPE can enter the nucleus of a cell. The present inventors have found that this fusion protein is useful in the treatment of c-Myc dependent cancer.

According to a first embodiment of the invention there is provided a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), said fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity in the nucleus.

According to a second embodiment of the invention, there is provided a pharmaceutical composition comprising a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), said fusion protein being capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus.

According to a third embodiment of the invention, there is provided a method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the step of subjecting to the cell a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), wherein the fusion protein is capable of penetrating the nucleus of the cell and inhibiting c-Myc activity within the nucleus.

According to a fourth embodiment of the invention there is provided a method of delivering a c-Myc inhibitor to the nucleus of a cell, the method comprising the steps of: fusing the c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus, and subjecting the fusion protein to the cell.

According to a fifth embodiment of the present invention, there is provided a method of making a chimeric fusion protein capable of penetrating the nucleus of a cell, the method comprising the step of fusing a c-Myc inhibitor to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE') to produce a chimeric fusion protein capable of penetrating the nucleus of a cell and inhibiting c-Myc activity within the nucleus.

According to a sixth embodiment of the invention, there is provided a method of preventing or treating a c-Myc dependent cancer in a subject, the method comprising the step of administering to the subject a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

According to a seventh embodiment of the invention there is provided a chimeric fusion protein for use in the prevention or treatment of a c-Myc dependent cancer in a subject, wherein the chimeric fusion protein comprises a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

According to an eighth embodiment of the invention, there is provided the use of a chimeric fusion protein comprising a c-Myc inhibitor fused to a genetically modified pseudomonas aeruginosa exotoxin a ('tPE'), in the manufacture of a medicament for the prevention or treatment of a c-Myc dependent cancer in a subject, the fusion protein being capable of penetrating the nucleus of a cell of the subject and inhibiting c-Myc activity within the nucleus.

The genetically modified pseudomonas aeruginosa exotoxin a ('tPE') can be in any suitable form so long as it is capable of being taken up into cells via endocytosis, as well as targeting and penetrating the nucleus of the cell. tPE may thus comprise one or more domains that enable it to translocate across cell membranes, including outer cell membranes and nuclear membranes. tPE may be produced in any suitable manner.

In some examples, tPE comprises domain Ia (amino acids 1-252 of mature lytic protein) or a biologically active fragment thereof directed against translocation across the nuclear membrane. In other examples, tPE comprises domain II (amino acids 253-364 of the mature cleavage protein) or a biologically active fragment thereof directed against translocation across the nuclear membrane.

In another example, tPE comprises domain Ia (amino acids 1-252 of the mature cleavage protein) or a biologically active fragment thereof and domain II (amino acids 253-364 of the mature cleavage protein) or a biologically active fragment thereof. In yet another example, tPE comprises domain Ia (amino acids 1-252 of the mature cleavage protein) fused to domain II (amino acids 253-364 of the mature cleavage protein).

Myc is a family of regulatory and proto-oncogenes that encode transcription factors, the best known example of which is c-Myc. Other examples of Myc are l-Myc and n-Myc. In cancer, c-myc is often constitutively (and possibly persistently) expressed, which in turn leads to increased expression of many other genes, some of which are thought to be involved in cell proliferation. Thus, without being bound by theory, it is believed that the overall expression of c-myc contributes to the development of cancer. Constitutive upregulation of the Myc gene has also been observed in cervical, colon, breast, lung and gastric cancers. In the human genome, c-myc is believed to regulate expression in about 15% of all genes by binding to a so-called enhancer cassette sequence (E-Box).

As used herein, the term "inhibitor" refers to a compound that is capable of inhibiting or blocking the activity of a particular target. These targets can be, but are not limited to, enzymes, receptors (neurotransmitters as non-limiting examples), proteins, genes, and any other molecule with a biological function. The various compounds and drugs are not limited to a single action and may therefore be considered inhibitors for a particular target even though they differ in structure. That is, inhibition against a particular target is a combined property of these compounds.

Thus, in one example, the inhibitors disclosed herein are c-Myc inhibitors. Any suitable type of c-Myc inhibitor may be used in conjunction with the subject matter names disclosed herein, provided that it is capable of inhibiting c-Myc in the nucleus, either directly or indirectly. The c-Myc inhibitor may be produced in any suitable manner.

In one example, the C-Myc inhibitor is fused to the C-terminus of tPE. In another example, the C-Myc inhibitor is fused to the C-terminus of domain II of tPE.

In some embodiments, the c-Myc inhibitor may be a peptide of any suitable sequence and length. In some embodiments, the c-Myc inhibitor may be a polypeptide of any suitable sequence and length. In some embodiments, the c-Myc inhibitor may comprise more than two or more peptides fused to tPE. The peptides may be the same or different from each other. In some embodiments, the c-Myc inhibitor may comprise more than two or more polypeptides fused to tPE. The polypeptides may be the same or different from each other. In some embodiments, the c-Myc inhibitor may comprise more than two or more peptides and/or polypeptides fused to tPE. The peptides and polypeptides may be the same or different from each other.

In some embodiments, the c-Myc inhibitor inhibits c-Myc, directly or indirectly. In some embodiments, the c-Myc inhibitor is capable of disrupting a c-Myc-dependent pathway in a c-Myc-dependent cancer. In some embodiments, the c-Myc inhibitor interferes with specific c-Myc DNA binding. In some embodiments, the c-Myc inhibitor blocks c-Myc/Max dimerization, thereby inhibiting transcriptional activation of c-Myc.

In another example, the c-Myc inhibitor is an H1 peptide derived from the carboxy region of helix 1(H1) of c-Myc, which may interfere with specific c-Myc DNA binding. The H1 peptide may have any suitable sequence and length, but is preferably H1(S6A, F8A) having the amino acid sequence NELKRAFAALRDQI (SEQ ID NO: 5). Other H1 c-Myc-inhibitory peptide sequences of interest include, for example, Omomyc (TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA; SEQ ID NO: 6).

As used herein, the terms "peptide," "protein," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogues, and it may be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that have been modified, either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a marker or bioactive component. The term peptide encompasses two or more naturally occurring or synthetic amino acids linked by a covalent bond (e.g., an amide bond). The amino acid residues are linked together by amide bonds. When the amino acid is an α -amino acid, an L-optical isomer or a D-optical isomer may be used, and the L-isomer is preferable in nature. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes, but is not limited to, modified sequences, such as glycoproteins. The term polypeptide is specifically intended to encompass naturally occurring proteins, as well as those produced recombinantly or synthetically. Substantially purified polypeptide, as used herein, refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. In one embodiment, the polypeptide is at least 50% (e.g., at least 80%) free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. In another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.

Conservative amino acid substitution representatives that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered 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).

Non-conservative amino acid substitutions may result from changes in: (a) the structure of the amino acid backbone in the substituted region; (b) charge or hydrophobicity of amino acids; or (c) the volume of the amino acid side chain. Substitutions that are generally expected to produce the greatest change in protein properties are those in which: (a) hydrophilic residues in place of (or substituted by) hydrophobic residues; (b) proline for (or by) any other residue; (c) a residue with a bulky side chain (e.g., phenylalanine) is substituted for (or by) a residue without a side chain (e.g., glycine); or (d) a residue having an electrically positive side chain (e.g., lysyl, arginyl, or histidyl) is substituted for (or by) an electrically negative residue (e.g., glutamyl or aspartyl).

A variant amino acid sequence may, for example, be 80%, 85%, 90% or even 95%, 98% or 99% identical to the native amino acid sequence. The procedures and algorithms for determining percent identity may be performed according to methods known in the art.

The cells may be isolated mammalian cells, such as cells cultured in vitro (cell culture) or cells obtained from a subject. In one example, the cell is a human cell. In another example, the subject is, but is not limited to, human, canine, porcine, bovine, murine, rodent, feline, primate (including non-human primate), and equine. That is, the treatment, exposure, contact, or administration of the chimeric protein to mammalian cells can be performed in vitro or ex vivo.

The mammalian cells may be of any suitable type. It may be a human cell, a primate cell, a cell of a laboratory animal such as a rodent or rabbit, a cell of a farm animal or a domestic animal such as a horse, sheep, goat or cow, or a cell of a companion animal such as a dog or cat.

Likewise, the subject may be a human, primate, laboratory animal, farm animal, livestock animal, or companion animal.

In some embodiments, the c-Myc-dependent cancer is a cancer or tumor of the cervix, colon, breast, lung, or stomach.

In some embodiments, the chimeric fusion protein may comprise a synthetic tag, such as a polyhistidine tag, HQ tag, HN tag, FLAG tag, or HAT tag or more thereof, for the purposes of protein production and purification. In some embodiments, the polyhistidine tag can be fused to the N-terminus of domain Ia tPE. The polyhistidine tag can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues in length.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier or one or more other ingredients.

The chimeric fusion protein or pharmaceutical composition (hereinafter "composition") can be administered to a subject in a prophylactically effective amount or a therapeutically effective amount as desired for the particular situation under consideration. The actual amount of the composition and the rate and timeline of administration of the composition will depend on the nature and severity of the cancer to be treated or the desired prevention. The prescription of a treatment, such as a dosage decision, will be within the skill of the medical practitioner or veterinarian responsible for the care of the subject. Typically, however, a composition for administration to a subject will comprise from about 0.01mg to 100mg of the compound per kg of body weight. In another example, the compositions disclosed herein are administered in an amount of about 0.1 to 10mg/kg body weight. In yet another example, the composition or compound will be at least 0.1mg/kg to 10mg/kg, 0.1mg/kg to 5mg/kg, 1mg/kg to 2.5mg/kg, 2.5mg/kg to 5mg/kg, 5mg/kg to 10mg/kg, 5mg/kg to 7.5mg/kg, 7.5mg/kg to 10mg/kg, at least 1mg/kg, at least 1.5mg/kg, at least 1.8mg/kg, at least 2mg/kg, at least 2.5mg/kg, at least 2.8mg/kg, at least 3mg/kg, at least 3.2mg/kg, at least 3.5mg/kg, at least 4mg/kg, at least 4.5mg/kg, at least 5.5mg/kg, at least 6mg/kg, at least 6.5mg/kg, at least 7mg/kg, At least 7.5mg/kg, at least 8mg/kg, at least 8.5mg/kg, at least 9mg/kg, at least 9.5mg/kg or at least 10 mg/kg.

In one example, the amount to be administered as described herein is to be understood as a daily administration regimen. In another example, the medicament will be administered to the subject daily, weekly, twice weekly (twice weekly), three times weekly, biweekly, monthly (i.e., once a month)), or any combination thereof. For example, the drug may be administered daily for the first week and twice weekly for the following 4 weeks. Alternatively, in another example, the drug may be administered to the subject twice a week for the first 2 weeks of treatment, and then once a month for another 3 months.

As used herein, the term "treatment" refers to any and all applications that treat a disease state or symptom, prevent the establishment of a disease, or in any way prevent, hinder, slow or reverse the progression of a disease or other undesirable symptom.

The term "treating" or "treating" as used herein is intended to mean providing a pharmaceutically effective amount of a peptide or its corresponding pharmaceutical composition or medicament, which is sufficient to act prophylactically to prevent the development of debilitating and/or unhealthy conditions; and/or providing the subject with a sufficient amount of the complex or a pharmaceutical composition or medicament thereof to reduce or eliminate the disease state and/or symptoms of the disease state as well as weakened and/or unhealthy conditions.

The fusion protein as described herein and above may be formulated into a composition suitable for administration, e.g., a pharmaceutical composition. When applicable, the peptide or protein may be administered with a pharmaceutically acceptable carrier. The "carrier" may include any pharmaceutically acceptable carrier so long as the carrier is compatible with the other ingredients of the formulation and not injurious to the patient. Pharmaceutical compositions for use may thus be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen. Thus, in one example, the present disclosure describes a pharmaceutical composition comprising, but not limited to, a peptide described herein, an isolated nucleic acid molecule for expressing the peptide, or a vector for amplifying the isolated nucleic acid molecule described herein. In another example, the disclosure describes an isolated nucleic acid molecule encoding a peptide as described herein. In another example, the disclosure describes a vector comprising an isolated nucleic acid molecule as described herein. In one example, the pharmaceutical composition comprises a peptide as described herein. In yet another example, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, vehicles, or carriers. Thus, in one example, a peptide as disclosed herein may further comprise a compound selected from, but not limited to, a pharmaceutically acceptable carrier, a liposomal carrier, an excipient, an adjuvant, or a combination thereof.

The composition, shape and type of dosage form of the peptides as disclosed herein will generally vary depending on the intended use. For example, a dosage form for acute treatment of a disease or related disorder may contain a greater amount of one or more active compounds than a dosage form for chronic treatment of the same disease. Similarly, a parenteral dosage form may contain a smaller amount of one or more active compounds than an oral dosage form used to treat the same disease or condition. These and other ways in which the particular dosage forms encompassed by the present invention differ from one another will be apparent to those skilled in the art. Examples of dosage forms include, but are not limited to: a tablet; a caplet; capsules, such as soft elastic gelatin capsules; a cachet; a lozenge; a lozenge; a dispersant; suppositories; an ointment; cataplasm (cataplasm); a paste; powder preparation; a dressing; a cream formulation; a cream medicament; a solution; a patch; aerosols (e.g., nasal sprays or inhalers); gelling; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms particularly suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a patient. Thus, in one example, the peptides disclosed herein are provided in a form selected from, but not limited to: tablets, caplets, capsules, hard capsules, soft elastic gelatin capsules, hard gelatin capsules, cachets, lozenges, troches, dispersions, suppositories, ointments, cataplasms, pastes, powders, dressings, creams, plasters, solutions, patches, aerosols, nasal sprays, inhalers, gels, suspensions, aqueous liquid suspensions, non-aqueous liquid suspensions, oil-in-water emulsions, water-in-oil liquid emulsions, solutions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution, or combinations thereof.

The composition may be administered to the subject in any suitable manner, including: parenteral, topical, oral, by inhalation spray, rectal, nasal, buccal, vaginal or via an implantable reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The pharmaceutically acceptable carrier may include any suitable diluent, adjuvant, excipient, buffer, stabilizer, isotonicity agent, preservative or antioxidant. It is understood that the pharmaceutically acceptable carrier should be non-toxic and should not interfere with the efficacy of the fusion protein. The exact nature of the carrier or any other additives of the composition will depend on the route of administration and the type of treatment desired. The pharmaceutical compositions may be prepared, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Sterile injectable forms of the compositions can be aqueous or oleaginous suspensions. These forms are known to those skilled in the art. For intravenous, cutaneous or subcutaneous injection, or injection at the site in need of treatment, the composition may be in the form of a parenterally acceptable aqueous solution having suitable pH, isotonicity and stability.

Orally acceptable dosage forms of the compositions include, but are not limited to, capsules, tablets, pills, powders, liposomes, granules, spheres, dragees, liquids, gels, syrups, slurries, suspensions and the like. Suitable oral forms are known to those skilled in the art. Tablets may include solid carriers such as gelatin or adjuvants or inert diluents. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. Such compositions and formulations typically contain at least 0.1 wt% of the chimeric fusion protein, and in one example, up to about 25 wt%, depending on its solubility in a given carrier.

The composition may be administered topically, particularly when the target of treatment includes an area or organ readily accessible by topical administration, including cancer of the eye, skin, or lower intestinal tract. The composition may be administered in the form of a solution, suspension, emulsion, ointment, cream, lotion, paste, gel, foam or aerosol. Suitable topical forms are known to those skilled in the art.

The composition may include a delivery vehicle for delivering the compound to a particular organ, tissue or type of cancer and/or for ensuring that the compound can be taken up, for example, through the skin or through the gut without loss of biological efficacy. The delivery vehicle may comprise, for example, lipids, polymers, liposomes, emulsions, antibodies, and/or proteins. Liposomes are particularly preferred for delivery of the compounds through the skin.

The compositions may be delivered using a sustained release system, such as a semipermeable matrix of a solid hydrophobic polymer containing the compound. Various sustained release materials are available and are well known to those skilled in the art. Sustained release capsules can release the compound for about 1 to 20 weeks depending on its chemical nature.

The composition may be administered to a subject with one or more other active agents to achieve an optimal prophylactic or therapeutic effect. The active agent can be, for example, alkylating agents, angiogenesis inhibitors, antiandrogens, antiestrogens, antimetabolites, apoptotic agents, aromatase inhibitors, cell cycle control agents, cell stressors, cytotoxins, cytoprotective agents, hormones, immunotherapeutic agents, kinase inhibitors, monoclonal antibodies, platinating agents, respiratory inhibitors, retinoids, signal transduction inhibitors, taxanes, and topoisomerase inhibitors.

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 this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention herein.

Definition of

As used herein, the following terms have their assigned meanings unless otherwise indicated.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including but not limited to glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. Standard single or three letter codes are used to denote amino acids.

A "fragment" is a truncated form of a native biologically active protein that retains at least a portion of its therapeutic and/or biological activity.

A "chimeric" protein contains at least one fusion polypeptide comprising a region in the sequence that is positionally different from the naturally occurring sequence. This region may typically be present in separate proteins and brought together in a fusion polypeptide; or they are normally present in the same protein, but in a new arrangement in the fusion polypeptide. For example, chimeric proteins can be produced by chemical synthesis or by generating and translating polynucleotides in which peptide regions are encoded in a desired relationship.

"conjugated", "linked" and "fused" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components by any means, including chemical conjugation or recombinant means.

In the context of polypeptides, a "linear sequence" or "sequence" is the sequence of amino acids in a polypeptide in the amino to carboxy terminal direction, wherein residues in the sequence that are adjacent to each other are contiguous in the primary structure of the polypeptide.

"recombinant" refers to the product of various combinations of in vitro cloning, restriction and/or ligation steps, as well as other processes to produce constructs that can be potentially expressed in a host cell.

As used herein, the term "PE" refers to pseudomonas aeruginosa exotoxin a ('PE'), which is a toxic virulence factor for pseudomonas aeruginosa bacteria. PE is expressed as a nascent protein with a length of 638 amino acids, but a highly hydrophobic leader peptide with 25 amino acids at its N-terminus is cleaved during secretion. PE contains different functions and domains. Following the leader peptide, PE has a receptor binding domain Ia (amino acids 1 to 252) consisting of an antiparallel beta sheet and a domain II (amino acids 253 to 364) with six consecutive alpha-helices, which enables PE translocation across the cell membrane, e.g., from the endoplasmic reticulum to the cytosol. Domain II is followed by domain Ib (amino acids 365-. The last 4 residues of domain Ib (amino acids 400-404) form together with domain III the catalytic subunit of the toxin with ADP-ribosyltransferase activity.