This Non-provisional application claims priority under 35 U.S.C. §119(a) on Patent Application No(s). 104139300 filed in Taiwan, Republic of China Nov. 26, 2015, the entire contents of which are hereby incorporated by reference.
The present invention relates to a recombinant cytotoxin, and especially relates to a programmed self-destruct cytotoxin, and particularly relates to a cytotoxin can result in apoptotic activity towards targeted cells and then the cytotoxin can be hydrolyzed and destroying itself.
A toxin is an organic or inorganic substance which, even at low concentrations, has a deleterious effect on living organisms. Many bacteria and higher plants produce cytotoxic proteins collectively called ribotoxins which function by being taken up by, and then inactivating the ribosomes of a target cell. The ribotoxins are considered to fall into two major classes: (1) NAD+-dependent ribotoxins, which appear to disable ribosomes by covalently attaching ADP-ribose to “elongation factor-2” protein; and (2) NAD+-independent ribotoxins, which appear to inactivate the 60S ribosomal subunit. It is the NAD+-independent ribotoxins and their derivatives to which the separation and purification methods of the invention apply. These ribotoxins affect only eucaryotic ribosomes which are lethal at low concentrations.
A ribotoxin may be a heterodimer or a polypeptide, wherein the heterodimer is formed by an enzymatically A chain polypeptide and a enzymatically inactive B chain polypeptide linked by disulfide bonds, and the non-catalytic B chain polypeptide binds to surface of a target cell to stimulate uptake of the ribotoxin into the cell. The heterodimer ribotoxin includes ricin, abrin, and modeccin. Other ribotoxins are single polypeptides which are cytoloxioally active, and are thus sometimes referred to as “A chain toxins” or “hemitoxins”.
Several ribotoxins, such as ricin and abrin, occur in nature in more than one form. Thus, these ribotoxins can be considered to represent several isotoxins, i.e. structurally similar proteins with quantitatively differing functional properties.
Some attempts have been made to take advantage of the cytotoxic properties of the ribotoxins by employing the unmodified polypeptides as therapeutic agents. However most efforts to use ribotoxins therapeutically have been focused on hybrid toxins, in which the cytotoxic moiety is covalently coupled to a “binding moiety” expected to bind specifically to certain cells, virus, or other macromolecules. The most common examples of hybrid toxins are immunotoxins, wherein the cytotoxic polypeptide is conjugated to a specific antibody; however, a variety of other binding moieties may be used. However, the hybrid toxins still would affect other normal periphery cells even if they have specificity.
In view of the above-mentioned problem, the present invention provides a novel recombinant cytotoxin. The recombinant cytotoxin of the present invention with apoptosis activity is able to kill target cells and does not affect the cells surrounding the target cells.
The present invention provides a recombinant cytotoxin comprising a cytotoxin, a penetrating peptide, and an Asp-Glu-Val-Asp (DEVD) sequence, wherein the DEVD sequence is inserted into the cytotoxin.
In one embodiment, the penetrating peptide is linked to the cytotoxin through a linker.
In one embodiment, the cytotoxin comprises ribotoxin, snake venom, bee venom, jellyfish venom or toad venom.
In one embodiment, the ribotoxin is selected from a group consisting of fungal-originated ribotoxin, ricin, abrin, emetine, diphtheria toxin, cinnamomin, camphorin.
In one embodiment, the fungal-originated ribotoxin comprises α-sarcin, gigantin, mitogllin, restrictocin, allergen, clavin or tricholin.
In one embodiment, the penetrating peptide comprises Tat, antennapedia or polyarginine.
In one embodiment, the DEVD sequence is inserted into the loop region of the ribotoxin.
In one embodiment, the DEVD sequence is inserted into the loop 2 of the ribotoxin.
In one embodiment, the Tat is inserted into the N-terminus of the cytotoxin.
The present invention also provides a pharmaceutical composition comprising the recombinant cytotoxin of the present invention and a pharmaceutically acceptable carrier.
The present invention further provides a use of the recombination cytotoxin of the present invention for preparing a pharmaceutical composition of treatment of cell proliferation disease.
In one embodiment, the recombinant cytotoxin is toptically administrated.
In one embodiment, the proliferation disease is cancer.
In one embodiment, the cancer comprises oral cancer, breast cancer, prostate cancer, leukemia, colorectal cancer, uterine cancer, ovarian cancer, endometrial cancer, cervical cancer, testicular cancer, lymphoma, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumors, skin cancer, stomach cancer, liver cancer, kidney cancer or nasopharyngeal cancer.
Detailed description of the invention is given in the following embodiments with reference to the accompanying drawings.
The present disclosure provides novel fusion proteins. Various aspects of the present disclosure relate to fusion proteins, compositions thereof, and methods for making and using the disclosed fusion proteins. By administrating the novel fusion proteins of the present invention to an organism, significantly increased positive response can be seen within the organism.
The following is a detailed description provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art would understand that modifications or variations of the embodiments expressly described herein, which do not depart from the spirit or scope of the information contained herein, are encompassed by the present disclosure. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the invention. The section headings used below are for organizational purposes only and are not to be construed as limiting the subject matter described.
The present invention provides a recombinant cytotoxin, a penetrating peptide, and an Asp-Glu-Val-Asp (DEVD) sequence, wherein the DEVD sequence is inserted into the cytotoxin.
Referring to
The term “toxin” as used herein, generally refers to specific, characterizable, poisonous chemicals, often proteins, with specific biological properties, including immunogenicity, produced by microbes, higher plants or animals.
The toxin includes any toxin substances produced by any living organisms (including bacteria and plants). The toxin includes, but is not limited to, ribotoxin, snake venom, bee venom, jellyfish venom or toad venom, preferably ribotoxin. The recombinant cytotoxin of the present invention can be produced using DNA recombinant technology. The cytotoxin can be covalently linked with another functional protein.
The term “ribotoxin” as used herein, generally refers to any peptide or polypeptide produced naturally or synthetically which is capable of targeting and enzymatically releasing a specific base located within a specific base sequence in a nucleic acid substrate. The ribotoxin includes, but is not limited to, fungal-originated ribotoxin, ricin, abrin, emetine, diphtheria toxin, cinnamomin and camphorin.
Further, the fungal-originated ribotoxin includes, but is not limited to, α-sarcin, gigantic, mitogllin, restrictocin, allergen, clavin and tricholin, preferably α-sarcin.
The term “cell penetrating peptide (CPP)” as used herein, generally refers to carrier peptide that is capable of crossing biological membrane or a physiological barrier. Cell penetrating peptides are also called cell-permeable peptides, protein-transduction domains (PTD) or membrane-translocation sequences (MTS). CPPs have the ability to translocate in vitro and/or in vivo the mammalian cell membranes and enter into cells, and directly carries an interestingly conjugated compound, such as a drug or marker, to a desired cellular destination, e.g. into the cytoplasm (cytosol, endoplasmic reticulum, Golgi apparatus, etc.) or the nucleus. Accordingly, the CPP can direct or facilitate penetration of an interesting compound across a phospholipid, mitochondrial, endosomal or nuclear membrane. The CPP can also directly carry an interesting compound from outside the cell through the plasma membrane, and into the cytoplasm or to a desired location within the cell, e.g., the nucleus, the ribosome, the mitochondria, the endoplasmic reticulum, a lysosome, or a peroxisome. Alternatively or in addition, the CPP can directly carry an interesting compound across the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal and/or pulmonary barriers.
Several proteins and their peptide derivatives have been found to possess cell internalization properties including but not limited to the Human Immunodeficency Virus type 1 (HIV-1) protein Tat (Ruben et al. J. Virol. 63, 1-8 (1989)), the herpes virus tegument protein VP22 (Elliott and O'Hare, Cell 88, 223-233 (1997)), the homeotic protein of Drosophila melanogaster Antennapedia (the CPP is called Penetratin) (Derossi et al., J. Biol. Chem. 271, 18188-18193 (1996)), the protegrin 1 (PG-1) anti-microbial peptide SynB (Kokryakov et al., FEBS Lett. 327, 231-236 (1993)) and the basic fibroblast growth factor (Jans, Faseb J. 8, 841-847 (1994)). A number of other proteins and their peptide derivatives have been found to possess similar cell internalization properties. The carrier peptides that have been derived from these proteins show little sequence homology with each other, but are all highly cationic and arginine or lysine rich. Indeed, synthetic poly-arginine peptides have been shown to be internalized with a high level of efficiency (Futaki et al., J. Mol. Recognit. 16, 260-264 (2003); Suzuki et al., J. Biol. Chem. (2001)).
The term “linker” as used herein, generally refers to a covalent bond, preferably a peptide bond. The recombinant cytotoxin may optionally include at least one linker. The linker is between the cytotoxin and cell penetrating peptide (CPP). In one embodiment, the linker comprises 1 to 5 amino acids.
In a specific embodiment, the cytotoxin is α-sarcin, cell penetrating peptide is Tat, and DEVD sequence is located at the loop region of α-sarcin, preferably loop 2 region, wherein the DEVD sequence more preferably is located at amino acid position 84 (Gly) of α-sarcin (
The present invention further provides a pharmaceutical composition comprising the recombinant cytotoxin and a pharmaceutically acceptable carrier.
The composition for treatment is formulated to be compatible with the route of administration. The composition can be formulated as a powder, a tablet, a pill, a granule, a capsule, a lotion, a suspension, a liposome formulation, a nasosphere, a patch, a suppository, an enema, a drip infusion, or an injection solution. The composition can be administered orally, intraarticularly, intraperitoneally, intrathecally, intrarterially, intranasally, intraparenchymally, subcutaneously, intramuscularly, intravenously, dermally, intrarectally, or topically.
The term “subject” as used herein, generally refers to human or non-human mammal, e.g. a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, or a primate, and expressly includes laboratory mammals, livestock, and domestic mammals. In one embodiment, the mammal may be a human; in others, the mammal may be a rodent, such as a mouse or a rat. In another embodiment, the subject is an animal model (e.g., a transgenic mouse model).
A solution for parenteral, intradermal, or subcutaneous administration can include: a sterile diluent such as water, saline, glycerin, fixed oils, polyethylene glycols, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl parabens; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent; or a buffering agent such as acetate or phosphate. The solution can be stored in ampoules, disposable syringes, or plastic or glass vials.
A formulation for injection or intravenous administration can include a carrier which is a solvent or a dispersion medium. Suitable carriers include water, physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) phosphate buffered saline (PBS), ethanol, polyols (e.g., glycerol, glycol, propylene glycol, and the like), and mixtures thereof. These compositions must be steriled and liquefied for injection. Fluidity of these compositions can be maintained with, for example but not limited, lecithin or a surfactant. Microbial contamination can be prevented by the inclusion of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Sugars and polyalcohols, such as manitol, sorbitol or sodium chloride, can be used to maintain isotonicity in the composition.
The present invention further provides a use of the recombinant cytotoxin for preparing a pharmaceutical composition of treatment of cell proliferation disease.
The recombinant cytotoxin of the present invention can pass across the membrane and penetrate into the cells to kill the cells. Next, the enzymes generated from apoptosis, such as caspase-3, can recognize the DEVD sequence on the recombinant cytotoxin and destroy the structure of the cytotoxin.
The term “cell proliferative disorders” as used herein, generally refers to disorders wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm to the multicellular organism. Cell proliferative disorders can occur in different types of animals and in humans. Cell proliferative disorders include, but are not limited to, cancers, blood vessel proliferative disorders, and fibrotic disorders, preferably cancer. The cancer includes, not is not limited to, oral cancer, breast cancer, prostate cancer, leukemia, colorectal cancer, uterine cancer, ovarian cancer, endometrial cancer, cervical cancer, testicular cancer, lymphoma, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumors, skin cancer, stomach cancer, liver cancer, kidney cancer or nasopharyngeal cancer, preferably oral cancer.
The recombinant cytotoxin can penetrate into the cell which stimulated to undergo the apoptotic pathway and cleaved by the enzyme generated from apoptotic pathway. Thus, the recombinant cytotoxin specifically affects the target cell, and does not affect the cells surrounding the target cell.
Additional specific embodiments of the present invention include, but are not limited to the following:
The gene fragment of α-sarcin protein was amplified from filamentous fungus Aspergillus giganteus using polymerase chain reaction (PCR). The gene fragment was ligated into pET22b plasmid to make a KZ-sarcin plasmid. Tat peptide was fused in the N-terminus of α-sarcin using pET28a/α-sarcin as a template for PCR amplification with two N-primers and C-primer. The N1-primer was 5′-NNNCCATGGGTAGAAAAAAACGAAGACAACGACGAAGAGGTGGTGGTAGC-3′ (SEQ ID NO: 1). The N2-primer was 5′-GACGAAGAGGTGGTGGTAGC gt-gacctggacctgcttgaacg-3′ (SEQ ID NO: 2). C-primer was
N1 primer carried the basic domain sequence of Tat peptide (MGRKKRRQRRR (SEQ ID NO: 4)) with linker (GGGS (SEQ ID NO: 5)) and NCO I site (underlined), N2 primer carried overlapping sequence of N1-primer (uppercase) and α-sarcin specific sequence (lowercase), and C-primer carried α-sarcin specific sequence (lowercase) with a NOT I site (underlined). After PCR amplification, the PCR products were digested by NCO I and NOT I restriction enzyme and ligated into pET22b plasmid to make a pET-22b/Tat-sarcin plasmid.
In the second-step of construction, two primers, the upper and lower primers, were used to insert a DEVD sequence (SEQ ID NO: 8) into the loop 2 of α-sarcin for amplification with pET-22b/Tat-sarcin plasmid as template. The upper primer was 5′-GACGAAGTGGATggcaagagtgatcactacctgctggag-3′ (SEQ ID NO: 6), carries a coding sequence of DEVD (uppercase) with corresponding sequence of α-sarcin (lowercase). The lower primer was 5′-cttgctgtgcttgggaggacg-3′ (SEQ ID NO: 7), carries α-sarcin specific sequence (lowercase) with corresponding sequence of α-sarcin. After PCR amplification, the PCR products were purified, self-ligated and transformed into the competent cells ECOS101 to obtain the pET22b/KZ-sarcin (pET-22b/Kazecin) plasmid.
Recombinant pET22b/KZ-sarcin plasmid was expressed in E. coli strain BL21 CodonPlu (DE3) in LB broth under IPTG induction at 37° C. for 2 hours. The culture medium was centrifuged to obtain bacteria pellet. The bacteria pellet was added to 50 mL lysis buffer and lysed by a sonicator. After high speed centrifugation at 39,000 g for 1 hour, the supernatant was removed to collect inclusion bodies. The inclusion bodies were lysed in denature binding buffer by sonicator. After high speed centrifugation, the supernatant was reacted with Ni+-His resin (Novagen) for 2 hours, washed with denature wash buffer and eluted with denature elute buffer to obtain KZ-sarcin recombinant protein (SEQ ID NO: 9). The KZ-sarcin recombinant protein (Kazecin) has the sequences as follows.
The KZ-sarcin recombinant protein (Kazecin) was confirmed by SDS-PAGE. KZ-sarcin was treated with caspase-3 (Sigma Chem. Co, U.S.A.) and PBS buffer at room temperature for 15 minutes, respectively, and then analyzed by SDS-PAGE. In control group, a mutant sarcin (Sarcin*) was used. As shown in
The rabbit reticulum lysate (RRL) was used in this Example to proceed to ribosome inactivation assay. The rabbit reticulum lysates (Promega Co.) were treated with the KZ-sarcin and analyzed by 1% agarose gel electrophoresis. Referring to
RRL translation system was used in this Example. The RRL was treated with KZ-sarcin at different concentration in solution containing 20 mM Hepes, 5 mM dithiothreitol, 5 mM magnesium acetate, 100 mM potassium acetate, 2 mM ATP, 0.4 mM GTP, 8 mM creatine phosphate, 50 mg/mL creatine phosphokinase, plus 20 μM amino acid mixture minus methionine, and 1200 Ci/mmol at 1 mCi/mL [35S]methionine. The cellular translation was initiated by additional 40 μg/mL luciferase mRNA at 37° C. for 90 min. The [35S] incorporated protein was TCA-precipitated, and collected by GF/A glass filter (Whatman Co.). The incorporation of [35S] radioactivity was counted in a liquid scintillation counter (Tri-Carb 2900TR). As shown in
KZ-sarcin or α-sarcin was chemically conjugated with fluophore Alexa-555. This was carried out by mixing 500 μg of KZ-sarcin or α-sarcin with 50 μg of fluophore Alexa-555 (Invitrogen Co.) in PBS to a final volume of 500 μL. HeLa cells were treated with serum free DMEM containing 2 μL of KZ-sarcin or α-sarcin at 37° C. for 1 hr. The mixture was washed with PBS solution and then observed by fluorescent microscopy and phase contrast microscopy. As shown in
In contrast, no fluorescent signal was observed in the cells that had been treated with fluorescent-labeled wild type α-sarcin (
293T cells (OD650=0.3) were grown in 50-mL flask in the presence of [35S]methionine (the final concentration of 3 mCi/mL; Amersham, U.S.A.). When the cell culture had attained 0.5 OD650 units, cell culture was treated with recombinant KZ-sarcin (1 μM). At various time intervals, the cells were taken and lysed immediately in 1 mL of ice-cold trichloroacetic acid (TCA). Peptides that incorporated with [35S]methionine were collected on Whatman GF/C filters. The incorporation of [35S] radioactivity was counted by scintillation counter (Tri-Carb 2900TR). In
HeLa cells grown on glass cover slides were washed twice with PBS and treated with serum free DMEM containing 2 μM of α-sarcin or KZ-sarcin (Kazecin) at 37° C. for 1 hour. After washing cells twice with PBS, the cells were incubated with Hoechst 33342 in serum free DMEM at 37° C. for 1 hour. At the removing the dye, the cells were fixed with 4% paraformaldehyde and visualized by fluorescent microscopy (Olympus FV1000). As shown in
Oral SAS cells were treated with KZ-sarcin and then collected at different time intervals of 1, 2, 3, 4 and 5 hours post-incubation. The cells were treated with trypsin at 25° C. for 30 minutes. After several thoroughly washings, cells were lysed with denaturation solution containing 1% SDS and analyzed on a SDS-PAGE, followed by western blotting analysis using anti-His antibody.
The SAS cells collected in Example 8 were lysed by a lysis buffer (50 mM HEPES, pH7.4, 25 mM CHAPS, 25 mM DTT). The cell lysates were incubated with caspase-3 substrate (Ac-DEVD-pNA) at 37° C. for overnight. The caspase-3 activity was measured at the absorbance at 405 nm using Ultrospec 3300 pro (Amersham Biosciences).
In this Example, as shown in
As mentioned above, the fungal-originated ribotoxin can inhibit the ribosomal protein synthesis, but cannot easily move into cells. In the present invention, a penetrating peptide such as Tat of HIV, is linked with the fungal-originated cytotoxin using the recombinant DNA technology so that the fungal-originated ribotoxin can penetrate a target cell (e.g., cancer cell) and kill the target cell.
However, the cytotoxin linked with cell penetrating peptide would kill not only the target cell, but also the periphery cells through utilization of conventional and existing techniques. In order to improve the problems, a specific sequence which can be recognized by caspase-3 (e.g., Asp-Glu-Val-Asp (DEVD)) is used to be inserted to the loop region of fungal-originated ribotoxins, particularly loop 2. In addition, the recombinant ribotoxins still maintain the activity of wild-type cytotoxin. Thus, the recombinant cytotoxins of the present invention only enter target cells, and could be cleaved by caspase-3 generated from apoptotic pathway of the target cells. Accordingly, the recombinant cytotoxins, for example but are not limited, KZ-sarcin of the present invention specifically target the cancer cells and do not affect the other normal cells.
In the description above, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. For example, well-known equivalent components and elements may be substituted in place of those described herein, and similarly, well-known equivalent techniques may be substituted in place of the particular techniques disclosed. In other instances, well-known structures and techniques have not been shown in detail to avoid obscuring the understanding of this description,
Although the present invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternative embodiments, will be apparent to persons skilled in the art. It is, therefore, contemplated that the appended claims will cover all modifications that fall within the scope of the present invention.
Number | Date | Country | Kind |
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104139300 | Nov 2015 | TW | national |