|Title of Invention||
RECOMBINANT CALCITONIN FUSED TO INTERLEUKIN-2
|Abstract||The present invention provides fusion proteins of Calcitonin with Interleukin 2 that have enhanced biological activity compared to Calcitonin. In particular, the present invention provides the process of preparing and using compositions of the fusion protein of Calcitonin with Interleukin 2. Also provided are calcitonin prodrugs and fusion proteins comprising IL2 and calcitonin that are designed to cleave and release calcitonin and/or interleukin-2 on administration and uses thereof. Compositions for treating cancer comprising said fusion proteins are provided.|
|Full Text||FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
THE PATENT RULES, 2003
(See Section 10; rule 13)
RELIANCE LIFE SCIENCES PVT.LTD.
an Indian Company having its Registered office at
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC, Thane Belapur Road,
Rabale, Navi Mumbai - 400 701
The following specificationtion describes and ascertains the nature of this invention and the manner in which it is performed: -
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of provisional application No. 973/MUM/2006, filed on June 21, 2006, the disclosure of which is incorporated by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to new biologically active fusion polypeptides, their preparation and pharmaceutical compositions thereof. More particularly, the present invention relates to recombinant polypeptides composed of one or more active parts derived from a natural or artificial polypeptide having a therapeutic activity. More particularly, the present invention relates to fusion proteins of Calcitonin and Interleukin and processes for preparation thereof. The present invention also relates to the use of the fusion protein of Calcitonin and Interleukin as an intermediate in the preparation of Calcitonin.
BACKGROUND OF THE INVENTION
Calcitonin is a small peptide produced by the parafollicular cells of the thyroid gland in mammals and by the ultimobranchial glands of birds and fish. Many types of Calcitonin have been isolated, such as human Calcitonin, salmon Calcitonin, eel Calcitonin, elkatonin, porcine Calcitonin, and chicken Calcitonin. There is significant structural non-homology among the various Calcitonin types. For example, there is only 50% identity between the amino acids making up human Calcitonin and those making up salmon Calcitonin. Historically, Calcitonin has been extracted from the Ultimobranchial glands (thyroid-like glands) of fish, particularly salmon.
During hypocalcaemia, Calcitonin reduces elevated plasma calcium concentration to normal levels by inhibiting bone resorption. Calcitonins are therefore used to treat a variety of conditions such as Paget's disease, post menopausal osteoporosis, bone metastasis and also to treat hypocalcemia resulting from vitamin D intoxication, neoplastic disease, thyrotoxicosis or hyperthyroidism. Salmon Calcitonin is a peptide hormone that decreases uptake of calcium from bone. When used to treat bone-related diseases and calcium disorders (such as osteoporosis, Paget's disease, hypocalcemia of
malignancy, and the like), it has the effect of helping maintain bone density. Porcine and human Calcitonins typically have an activity of 100 to 200 IU/mg and salmon Calcitonin typically has an activity of up to 6500 IU/mg.
Human Calcitonin (hCT) is a peptide hormone containing 32 amino acid residues which is produced primarily by the Parafollicular (also known as C) cells of the thyroid. Salmon calcitonin is a polypeptide with a molecular weight of 3431.9, which consists of 32 amino acids. It has a disulphide bridge (cystine link) between the first and seventh amino acids at the amino-terminal end of the polypeptide chain, the disulfide bridge being essential for its biological activity, and a prolinamide group at the carboxyl terminal amino acid. The presence of this disulphide bridge contributes to the lack of stability of Calcitonin because, under thermal stress, it is susceptible to beta-elimination to produce free thiols. These thiols render the molecule vulnerable to degradation via various pathways and may also increase the occurrence of disulphide bond interchanges thus affecting the conformation and consequently the activity of the polypeptide.
The primary, secondary and tertiary structures of proteins and polypeptides are all vulnerable to various types of disruption. Some proteins and peptides are physically unstable as a result of, for example, adsorption, aggregation or denaturation. Others are chemically unstable as a result of, for example, oxidation, hydrolysis, deamidation, beta-elimination, racemisation or disulphide exchange (if the polypeptide contains a disulphide bridge e.g. a cystine link). Many proteins and polypeptides are susceptible to a number of these factors.
Calcitonins are currently only available in solution and are administered by intravenous infusion, by intramuscular injection, subcutaneously or intranasally. In order to maintain biological activity pharmaceutical preparations containing calcitonin must be stored at a temperature of 2 to 8°C. Storage at low temperatures is necessary for slowing down degradation, which occurs at a high rate in the liquid phase.
Salmon calcitonin has usually been administered by injection or by nasal administration. However, these modes of administering calcitonin are significantly less convenient than oral administration and create more patient discomfort. Often this inconvenience or discomfort results in substantial patient noncompliance with a treatment regimen.
Bioavailability following subcutaneous and intramuscular injection in humans is high and similar for the two routes of administration (71% and 66%, respectively). However, reproducible blood levels of peptides such as salmon calcitonin are difficult to achieve when administered orally. This is believed to be because these peptides lack sufficient stability in the gastrointestinal tract, and tend to be poorly transported through intestinal walls into the blood. Calcitonin has short absorption and elimination half-lives of 10-15 minutes and 50-80 minutes, respectively. Salmon calcitonin is primarily and almost exclusively degraded in the kidneys, forming pharmacologically inactive fragments of the molecule.
Proteolytic enzymes of both the stomach and intestines may degrade salmon calcitonin, rendering it inactive before the calcitonin can be absorbed into the bloodstream. Any salmon calcitonin that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas (typically having neutral to basic pH optima). Other difficulties arising from the oral administration of salmon calcitonin involve the relatively large size of the molecule, and the charge distribution it carries. This may make it more difficult for salmon calcitonin to penetrate the mucus along intestinal walls or to cross the intestinal brush border membrane into the blood. These additional problems may further contribute to the limited bioavailability of salmon calcitonin.
Being a small peptide, the process of isolation of this protein has posed difficulties. Previous attempts were made in terms of making a oligomer or multimers of calcitonin. Calcitonin is difficult to express in E. coli in recombinant form due to its small size. The options to overproduce calcitonin is to make concactamers of this with N-terminal
methionine, which again has its drawback as cyanogen bromide would have to be used to cleave at the N-ternminal methionine or to express it as a fusion with some other protein. (Sinko et.al , 1995 J.Pharm. Sci. 1995: 84:1374-1375; Hastewell et.al. 1992, Clin. Sci. 82:589-594; Beglinger et.al. 1992, Eur J. Clin. Pharmacol 43:527-531; Antonim et.al. 1992, Clin. Sci. 83:^27-631; Gras & Wittip, 1996, J.Am. Chem. Soc. 83:1510)
Large- scale preparation of recombinant human calcitonin from a multimeric fusion protein produced in E. coli is described by H. Ishikawa.(Journal of Bioscience and Bioengineering volume 87:, Issue 3 page 296-301, 1999). The multimeric fusion protein has a drawback in that cyanogen bromide was used to cleave the multimeric fusion protein into individual entities, Cyanogen bromide being a potentially hazardous chemical limits its use. (Environmental health & safety USA, MSDS number C6600)
Liquid compositions and methods for stabilization for oral delivery of human calcitonin are described in PCT publication WO 9800155. An aqueous liquid composition for stable storage of human calcitonin comprises an aqueous mixture of SDS and an organic acid. A nonaqueous liquid composition for stable storage of human Calcitonin comprises about 90-100 % by volume of a mixture of C8/C10 mono- and di-glycerides and about 0-10 % by volume of a polar, nonaqueous solvent. Both of these stabilized human Calcitonin formulations provide significant intestinal absorption of Calcitonin.
The Italian patent 1259140 relates to pharmaceutical compositions mainly comprising a polypeptide, for example Calcitonin, and co-freeze-dried lysogangliosides for oral administration in the form of gastric-protected capsules and/or tablets so as to promote absorption and bioavailability of the Calcitonin without creating biological imbalances in the organs and tissues which come into contact with the compounds used in the formulations.
PCT publication WO2004012772 describes a method of orally administering pharmaceutical compositions comprising Calcitonin in combination with oral delivery agents, prior to the consumption of food in humans; a method of treatment of disorders
responsive to the action of Calcitonin employing such method of administration; and also oral Calcitonin pharmaceutical compositions with particular ratios of the amount of oral delivery agent to the amount of Calcitonin which include N- (5-chlorosalicyIoyl)-8-aminocaprylic acid (5-CNAC),N-0-[2-hydroxybenzoyl] auminodecanoic acid (SNAD) and N- (8- [2-hydroxybenzoyl] amino) caprylic acid (SNAC), disodium salts and hydrates and solvates thereof.
A solid pharmaceutical composition suitable for the oral delivery of a pharmacologically active agent comprising the peptide calcitonin, crospovidone or povidone, and 5-CNAC is described in NZ526196. The composition can be used in the manufacture of a medicament for the treatment of a bone related disease or calcium disorder such as osteoporosis.
Oral bioavailability of salmon calcitonin (sCT), which is a peptide is extremely low due to degradation in the gastrointestinal tract (GIT) and low epithelial permeability. Attempts to enhance oral delivery were made by the use of enzyme inhibitors and absorption enhancers., such as ovomucoids and glycyrrhetinic acid which have been investigated as protease inhibitor and absorption enhancer, respectively. Enzymatic degradation revealed that sCT is degraded extensively by intestinal serine proteases such as trypsin, a-chymotrypsin, and elastase. Various ovomucoid (OVM) species such as chicken, duck and turkey ovomucoid (tOVM) were investigated for their inhibitory action. Duck and turkey ovomucoids stabilized sCT against degradation in the presence of the proteases for an hour. The permeability of sCT was enhanced in the presence of glycyrrhetinic acid. Regional permeability in rat GIT revealed that sCT is permeated mostly from ileum followed by jejunum, colon, stomach and duodenum. Therefore, the formulation of sCT was targeted to jejunum. (Shah et.al. J. Pharm Sci. 2004, 93:392-406; Agarwal et.al J. Pharm. Pharmacol. 2001, 53:1131-1138; Kompella et.al Adv. Drug Delivery Rev. 2001:46:211-245)
An osmotically controlled bilayered tablet coated with enteric polymers was successfully
used to prepare sCT formulation. Dissolution studies were performed for a period of 4hrs that showed dual controlled release of the drug and the inhibitor. Characterization of sCT in the formulation using DSC, FT-IR, powder X-ray diffraction and gel electrophoresis studies revealed that the structure was conserved after subjecting to formulation conditions. A seven-factor, three-level optimization design was used to evaluate the effect of critical process variables including the orifice size, coating level, amounts of sodium chloride, Polyox N10, Polyox N80, Carbopol 934P, Carbopol 974P.
Many proteins and polypeptides have potential as pharmaceutical agents but because they are susceptible to both physical and chemical degradation they are often too unstable to be included in pharmaceutical formulations. In particular such proteins and polypeptides do not have adequate shelf life.
Although the use of excipients to stabilize proteins and polypeptides has proved suitable for the stabilization of some proteins and polypeptides it is inadequate for the stabilization of less stable proteins and polypeptides, particularly those containing a disulphide bridge, such as a cystine link, e.g. calcitonin. There is therefore a need for further methods of stabilizing proteins and polypeptides, which are capable of stabilising these less stable proteins and polypeptides. In particular there is a need for a method of stabilising polypeptides with a disulphide bridge, e.g. calcitonin.
It is widely recognized in the pharmaceutical industry that oral drug delivery is the preferred mode of drug administration. Oral administration is simpler than other invasive methods of administration. Oral administration is generally more acceptable to patients and so increases patient compliance. Oral administration also avoids the need to use sterilized equipment such as syringes when administering the pharmaceutical, which results in increased safety for the patient.
In contrast to oral pharmaceutical formulations injectable pharmaceutical solutions must be prepared in sterile conditions in highly regulated laboratories. This is necessary because pharmaceuticals administered by injection are delivered directly into the blood
stream or the muscles of the patient, so even a small amount of contamination could cause significant adverse effects. Pharmaceuticals, which are administered in oral dosage forms, are ingested and pass through the alimentary canal before the active component is released into the blood stream or into the tissues of the patient. Thus the body will excrete small amounts of contamination during the normal digestive process. The requirement to prepare pharmaceutical solutions under highly sterile conditions increases the cost and inconvenience of their preparation.
Although oral dosage forms are desirable, their provision is not always possible. In the case of polypeptides such as Calcitonins, the provision of oral dosage formulations is hindered by the high instability of the polypeptides. These materials are not suitable for processing into a oral dosage forms because they cannot withstand the physical and chemical stresses of conventional formulating techniques. There is therefore a need for Calcitonin in a solid dosage form; more particularly there is a need for a solid oral dosage form of Calcitonin. There is a farther need for Calcitonin in a solid dosage form with an improved shelf life, more preferably one that will not have to be stored at low temperatures.
There is a further need for solid dosage formulations of proteins or polypeptides in which the protein or polypeptide is homogeneously distributed throughout the formulation. This allows an accurate amount of pharmaceutical to be administered, which is particularly important for potent pharmaceuticals, such as Calcitonin, in which any deviation from the desired dose would be significant in its effects. There is also a need for solid compositions containing a homogeneous distribution of Calcitonin, which are-useful to prepare the solid dosage formulations described above.
Scientists recently found that 2 immune-system proteins - interleukins IL-2 and IL-12 effectively fight oral tumors in mice, and scientists believe that this therapy holds great promise in the treatment of head and neck squamous-cell carcinoma in humans With around 200 different types of cancer affecting 4 out of 10 people at some point in their lives, finding an effective treatment for an individual's particular cancer is no mean feat.
Oral cancer has a higher proportion of deaths per number of cases than breast cancer, cervical cancer and skin melanoma and there has been little improvement in survival rates over the past 30 years. Chemotherapy and radiotherapy are now widely known as treatments for some forms of the disease, but little is known about the other treatments on offer and even less about cancer treatments in development.
Calcitonin also has been found to be present in a variety of cancer treatments. Calcitonin inhibits invasion of breast cancer cells.( Bo Han et.al. Int. J. Oncol. 2006, 28: 807-814). The analgesic effect of Calcitonin has been demonstrated by Kimena et. Al (Gan No Rinsho 1987, July 33:921-927). Various studies on Calcitonin in relieving pain in cancer has been studied. ( Gennaric, et.al. Curr. Ther. Res. I988 44:712-722; Int. J. Clin. Pharmacol Ther. Toxicol 1987 : 25, 229-232; Roth A. et.at.,. Oncology 1986: 43: 283-287; Gennari et.al. Curr. Ther. Res. 1989: 45:804-812 ) A doubled blind controlled trial for salmon Calcitonin in pain due to malignancy has been demonstrated by Hindley et.al. cancer-chemother phamocaon 1982:9:71:74)
OBJECT OF THE INVENTION
It is the object of present invention to provide a novel fusion protein of Calcitonin and Interleukin.
It is the object of the present invention to provide a process for preparation of the fusion protein of Calcitonin and Interleukin.
It is the object of the present invention to provide compositions of the of the fusion protein of Calcitonin and Interleukin.
It is the object of the present invention to provide the fixed protein for use in therapies such as cancer particularly breast cancer and for calcitonin related disorders or deficiencies.
It is also an object of the present invention to provide an efficient process for isolating Calcitonin by cleaving the fused protein.
SUMMARY OF THE INVENTION
The present invention is directed to a first polypeptide (IL-2) covalently linked to a second polypeptide (Calcitonin).
In one embodiment, the human interleukin-2 has the sequence consisting of: MPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIISTLT [SEQIDNO 1]
In one embodiment, the human calcitonin has the sequence consisting of: CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPG [SEQ ID NO 2]
In one embodiment the polypeptide is expressed in the form A-L-B which is prepared via recombinant DNA technology, wherein A= Interleukin 2, L- linker, B- calcitonin. In a particular embodiment, the linker has the sequence of Asp4-Lys (D4K).
According to a preferred form of this embodiment, the sequence of the polypeptide is: MPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIISTLTDDDDKCGNLSTCMLGTYTQDFNKFHTFPQTAIG VGAPG[SEQ ID N0 3]
In one embodiment of the invention, the fusion protein comprises IL-2- CT. According to a preferred form of this embodiment, the sequence of the polypeptide is: MPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIISTLTCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPG. [SEQ ID NO 4]
In another embodiment, an IL2-CT fusion protein may comprise one or more IL-2 units fused to one or more CT units thus allowing different doses of IL-2 and CT to be available in the formulation. In some embodiments, several unit CT sequences are fused to each IL-2 peptide sequence as IL-2 is more toxic that CT. In some embodiments each unit sequence of IL-2 and CT is separated from one another by an enterokinase-sensitive D4K linker peptide.
In another embodiment, the present invention also provides isolated DNA sequences encoding one or more of the fusion proteins described above.
In yet another preferred embodiment, the present invention provides recombinant expression vectors comprising such DNA sequences, host cells containing the expression vectors, and processes for producing the recombinant fusion proteins by culturing the host cells.
In another preferred embodiment the present invention also provides pharmaceutical compositions comprising a purified fusion protein as described above and a suitable diluent, carrier, or excipients are also provided by the present invention.
In another preferred embodiment the present invention provides a fusion protein, which are useful in therapy, diagnosis and assays for conditions mediated by Calcitonin.
In another preferred embodiment the present invention provides a fusion protein, which are useful in therapy, diagnosis and assays for conditions mediated by Interleukin and Calcitonin.
The present invention also relates to novel compositions, in particular to compositions comprising Calcitonin or a fragment or conjugate thereof and to methods for preparing such compositions. It also relates to oral formulations comprising the compositions and to shelf stable Calcitonin or a fragment or conjugate thereof.
The present invention also relates to oral pharmaceutical formulations chosen from the group consisting of tablets, minitablets, capsules, granules, pellets, powders, effervescent solids, and chewable solid formulations, said formulation comprising a fusion peptide comprising calcitonin or a conjugate thereof and interleukin-2.
The present invention provides methods for preparing a fusion protein of Calcitonin and Interleukin-2 which comprises one or more of the following steps:
1. Cloning of IL-2 with vectors.
2. Cloning of Calcitonin with vectors.
3. Fusion of cloned IL-2 with cloned Calcitonin.
4. Cloning of fused IL-2 - Calcitonin into vectors.
5. Expression of Fused IL-2 - Calcitonin.
6. Studies of expression.
7. Culturing of fused protein in fermentor.
8. Purification of the fused IL-2- Calcitonin protein.
9. N- terminal sequencing of the fused IL-2 - Calcitonin protein.
10. Bioassay/ Toxicity studies
11. Study of absorption of IL-2-CT.
The present invention further provides an improved process for isolating Calcitonin by using the fused protein of IL-2- Calcitonin which comprises one or more of the following steps:
1. Cloning of IL-2 with vectors.
2. Cloning of Calcitonin with vectors.
3. Fusion of cloned IL-2 with cloned Calcitonin.
4. Cloning of fused IL-2 - Calcitonin into vectors.
5. Expression of Fused IL-2 - Calcitonin.
6. Studies of expression.
7. Culturing of fused protein in fermentor.
8. Purification of the fused IL-2- Calcitonin protein.
9. Cleavage with enterokinase
10. Isolation of Calcitonin.
The present invention provides a process to produce Calcitonin in a pure form, in the form of fusion protein, to protect it from degradation by native endoproteases of E. coli. After purification it is intended to cleave the fusion partner leaving Calcitonin intact.
In one embodiment the present invention also aims to present the fusion protein as a prodrug of Calcitonin, which will be used for oral delivery of Calcitonin. Calcitonin is not absorbed orally due to the attack of proteolytic enzymes. It is absorbed in the lower intestine. Hence there is a need for stabilising CT by delivery as a prodrug: The fusion protein has a linker, which can be cleaved by enterokinase present only in the intestine of the body. Hence this fusion protein functions as a prodrug for the oral delivery of calcitonin.
Interleukin-2 is a hormone that is naturally produced in the body. When administered as part of an immunotherapy program, IL-2 uses the body's own defense system to recognize and destroy cancer cells. Specifically, IL-2 plays a major role in immune regulation because it stimulates the proliferation of activated T lymphocytes. There is no easy way to explain this phenomenon. The mucosal immune system is known to be the site of priming for two paradoxically opposite purposes, i.e., tolerance and mucosal immunity. The usual response of the gastrointestinal tract to antigens is tolerance rather than immunity (Chen et al., 1995). The mechanisms of oral tolerance remain unclear.
The present invention provides a fusion protein for potential therapy in cancer. Calcitonin of late has gained importance in breast cancer. IL-2 is also indicated in cancer, hence the fusion protein of IL2- CT can be used in cancer.
BRIEF DESCRIPTION OF THE FIGURES
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fig.l: illustrates PCR amplification of IL-2 with signal sequence from the cDNA obtained from RNA of Jurkat cells wherein: Lane 1 and 2 : pfu amplified IL2 Lane 3 and 4: Taq amplified IL-2 Lane 5: 1 kb ladder
Figure 2 illustrates Nde- BamHl digestion of IL-2/pGEMT clones wherein 7 clones
digested with Nde-BamHl released the IL-2 fragments, and wherein:
Lanes 1-7 represent clones 1-7 respectively.
Lane 8 is Calcitonin
Lane 9 is 100 base pair ladder
Lane 10 is 1 kb ladder
Figure 3 : illustrates the restriction digestion of pET24 A/IL-2 wherein: Lanes 1-8 show pET 24A/IL-2 digested with Nde-1 and BamH-1 clones 1-8 respectively. Lane 9 is 100 bp ladder
Lane 10-11 is pET24/IL-2 digested with Nde-1 and BamHl of clones 9 and 10 respectively. Lane 12 is lkb ladder.
Figure 4 illustrates the PCR products of IL-2 and Calcitonin wherein the lanes are
described as below:
Lanes 3, 4 represent Calcitonin
Lanes 5, 6- represent IL-2
Lanes 7 is 100 bp ladder
Lane is 1 kb ladder
Figure 5 illustrates the gradient PCR for amplifications of Calcitonin after annealing of the oligonucleotides at following temperatures: 50.4°C, 52.7 °C, 55.1 °C , 57.5 °C.
Figure 6 illustratesPCR amplification of Calcitonin for cloning wherein the
Lanes 1-5 indicate the PCR amplified Calcitonin product Lane 6 indicates the lOObp ladder
Figure 7 illustrates PCR to generate IL-2- CT fusion wherein the Lane 1 is the fusion product and Lane 8 is 100 bp ladder and Lane 9 is 1 kb ladder.
Figure 8 illustrates Nde-Bam Digestion of IL2 -CT pGEMT clone wherein the Lanes 1-4 represent the clones 1-4 respectively.
Figure 9 illustrates Nde-Bam Digestion of IL2-CT/ pET24A clone wherein the Lanes 1 -6 represent clones 1 -6 respectively Lanes 7 represents 1 kb ladder Lane 8 represents lOObp ladder
Figure 10 illustrates SDS PAGE profile of IL-2-CT fusion protein expressed from BL21 DE-3 Host cells wherein the lanes represent the post induction at 0, 1, 2, 3,and 5 hours respectively.
Figure 11 illustrates wash optimization of inclusion body
Figure 12 illustrates the Final Inclusion body wherein lane 1 is standard IL-2 and the lane 2 is fusion protein
Figure 13 illustrates the refolded IL2-CT wherein Lane 1 represents marker Lane 2 represents solubilised Inclusion body Lane 3 represents refolded IL2-CT (0.5 mg/ml) Lane 4 represents refolded IL-2 -CT (0.2 mg/ml)
Figure 14 illustrates the plasmid DNA containing IL-CT. The DNA was sequenced with M-13 universal primers in both the directions.
Figure 15 illustrates the sequence of the recombinant Calcitonin fused to Interleukin
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides a fusion protein for potential therapy in cancer. Calcitonin of late has gained importance in breast cancer. IL-2 is also indicated in cancer, hence the fusion protein of IL2- CT can be used in cancer.
Since Interleukin has a potential to stabilize peptides, the present invention provides a stabilized Calcitonin wherein Interleukin has been used as fusion expression partner.
Further as Calcitonin has recently been indicated for cancer, the present invention aims to combine Interleukin and Calcitonin and present as fusion protein, which can be suitable for formulations not limited to oral delivery and has enhanced activity. The fusion protein is able to provide the therapeutic effects of both Interleukin and Calcitonin. Definitions:
As used herein the term "interleukin-2" or "IL-2" means IL-2 polypeptides and analogs thereof having substantial amino acid sequence identity to wild type mature mammalian IL-2s.
As used herein the term "Calcitonin" or "CT" and analogs can be selected from human, eel, salmon.
The term "fusion protein", "IL2-CT" refers to the fused Interleukin-2 with recombinant Calcitonin with a linker. As used herein, an IL2-CT fusion protein may comprise one or more IL-2 units fused to one or more CT units thus allowing different doses of IL-2 and CT to be available in the formulation. In some embodiments, concatemers of several unit CT sequences are fused to a single IL-2 peptide sequence as IL-2 is more toxic that CT.
The present invention provides fusion proteins of Calcitonin with Interleukin 2 and the resultant fusion protein have enhanced biological activity compared to Calcitonin. In
particular the present invention provides Calcitonin with enhanced biological activity, which renders them applicable for oral delivery.
As used herein a spacer or linker peptide is inserted between the Calcitonin and Interleukin. The spacer or the linker peptide is preferably non-charged and more preferably non-polar or hydrophobic. The length of a spacer or linker peptide is preferably between 1-100 amino acids, more preferably between 1 and about 50 amino acids, or between 1 and about 25 amino acids and even more preferably between 1 and about 15 amino acids, and even more preferably less than 10 amino acids. In one embodiment the spacer contains a sequence of 4 Aspartic acid and 1 Lysine amino acids, which is, indicated as D4K further herein the present invention. In yet another embodiment, the linker contains a motif that is recognized by a site-specific cleavage agent. In another alternative embodiment of the invention, the fusion protein i.e. Calcitonin and IL-2 are separated by a synthetic spacer that is preferably non-charged, and more preferably non-polar and hydrophobic.
PREPARATION OF FUSION PROTEINS:
Non-limiting methods for synthesizing useful embodiments of the invention are described in the examples herein as well as assays for testing the properties for in vitro activity and pharmacokinetics and in vivo activities in animal models.
In one embodiment, the synthesis of Calcitonin and Interleukin-2 fusion peptides may follow the stepwise solid phase strategy reported in Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154, the teachings of which are incorporated herein by reference. The present invention provides a method for preparation of the fusion protein of Calcitonin with Interleukin-2, which comprises of the following steps:
1. Cloning of IL-2 in cloning vectors.
2. Cloning of Calcitonin in cloning vectors.
3. Fusion of IL-2 with Calcitonin.
4. Amplification of fused product by PCR.
5. Cloning of fused IL-2 - Calcitonin into cloning vectors.
6. Cloning of fused IL-2 - Calcitonin into expression vectors
7. Studies of expression.
8. Culturing of fused protein in fermentor.
9. Purification of the fused IL-2- Calcitonin protein.
10. N- terminal sequencing of the fused IL-2 - Calcitonin protein.
11. Bioassay/ Toxicity studies
12. Study of absorption of IL-2-CT.
The present invention further provides an improved process for isolating Calcitonin by using the fused protein of IL2- Calcitonin which comprises the following steps:
1. Cloning of IL-2 gene with vectors.
2. Cloning of Calcitonin gene with vectors.
3. Fusion of IL-2 with Calcitonin coding sequences with a D4K peptide coding sequence linker.
4'. Amplification of fused product 6y PCR
5. Cloning of fused IL-2 - Calcitonin into vectors.
6. Expression of Fused IL-2 - Calcitonin.
7. Studies of expression.
8. Culturing of fused protein in fermentor.
9. Purification of the fused IL-2- Calcitonin protein.
10. Cleavage with enterokinase
11. Isolation of Calcitonin.
12. Amidation of Calcitonin
13. N- terminal sequencing of the Calcitonin protein.
In a preferred embodiment, IL2-CT nucleic acids encoding IL2-CT fusion proteins are used to make a variety of expression vectors to express IL2-CT fusion proteins. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the IL2-CT fusion protein. The term "cortroll sequences" refers to DNA
sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the IL2-CT fusion protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the IL2-CT fusion protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters,
which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences that flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
The IL2-CT fusion proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a IL2-CT fusion protein, under the appropriate conditions to induce or cause expression of the IL2-CT fusion protein. The conditions appropriate for IL2-CT fusion protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect, plant and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Saccharomyces cevevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, CI29 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human cells and cell lines.
In a preferred embodiment, the IL2-CT fusion proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenylation signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, are well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In a preferred embodiment, IL2-CT fusion proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase
and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the IL2-CT fusion protein in bacteria. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes that render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
In one embodiment, IL2-CT fusion proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
In a preferred embodiment, IL2-CT fusion protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorphs Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
In one embodiment, the IL2-CT nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or
heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the IL2-CT nucleic acids, proteins and antibodies at any position. For example, the label should be capable of producing, either directly or indirectly, a detectable signal. The detectable moiety may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al, J. Immunol. Meth, 40:219 (1981); and Nygren, J. Histochem. and Cytochem, 30:407 (1982).
Among the many systems available for heterologous protein production, E. coli is a preferred host because of its short doubling time, high-density growth on inexpensive substrates, its well-characterized genetics, a number of available host strains. Generally proteins produced in E. coli are of full length and in a biologically active form.
In recent years, the pET vectors (commercialized by Novagen, Madison) have gained increasing popularity. In this system, target genes are positioned downstream of the bacteriophage T7 late promoter on medium copy number of plasmids, the highly processive RNA polymerase is supplied in trans, typically host strains contain a prophage encoding the enzyme under the control of lac UV5. This system tends to overproduce proteins of interest but this too has its shortcomings. High levels of mRNA can cause ribosome destruction and ultimately cell lysis. Sometimes even empty pET plasmids can be toxic to E. coli and leads to cell death.
Another limitation of strong promoters like T7 is that the protein produced fails to fold back into its native conformation and generally tends to aggregate as inclusion bodies. cAMP deficient BL21(DE3) cells can be used for strain selection for fermentation or large scale processing. Although, glycosylation is not possible in E. coli, yet it is a robust strain for the cost effective production of eukaryotic proteins.
Earlier fusion proteins were made to facilitate easier purification of proteins by using affinity columns onto which the fusion partner would bind and thus help in the recovery of the protein of interest from the general pool of host proteins. The present invention generates fusion proteins with varied applications. It could improve the solubility of the passenger protein or it could lend to better stability of the protein. The purification can be performed without ion exchange processes.
Although commercial vectors are available with various tags, each has its inherent pros and cons. To overcome this problem, the present invention aims to fuse Calcitonin to Interleukin 2, so that the size of Calcitonin increases while at the same time, IL-2 can provide stability to the molecule.
The present invention provides vectors for cloning of IL2, Calcitonin and for the fused IL-2 -Calcitonin. The vectors can be selected from pUC19, pGEMT, pBScript. In the preferred example pGEMT vector was used for cloning.
In the preferred example the nucleic acid encoding the fusion protein is transfected into the host cell using the recombinant DNA technology. In the context of the present invention the foregoing DNA includes a sequence encoding the fusion proteins of the present invention. Suitable host cells include prokaryotic, yeast or higher eukaryotic, XL-1, ToplOF', DH 5alpha, JN 109, BL21DE3 and others know in the art. In the preferred examples XL-1 , ToplOF' and BL21 DE-3 was used as host.
The present invention also provides a process for preparing the recombinant proteins of the present invention including culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes the fusion protein of the present invention under conditions that promote expression. The desired fusion protein is then purified from culture media or cell extracts.
As used herein, the expression vectors are capable of expressing fusion proteins composed of Interleukin-2, a linker composed of (Asp)4-Lys (D4K) and Calcitonin. The
D4K peptide is a target for cleavage by the protease enterokinase. (LaVallie E R, Rehemtulla A, Racie L A, DiBlasio E A, Ferenz C, Grant K L, Light A, McCoy J M. J Biol Chem. 1993; 268:23311-23317). The produced fusion proteins allows Calcitonin and IL-2 to be separated through digestion with enterokinase during purification.
DNA sequences encoding the polypeptides of the fusion partners and a specific amino acid sequence for enzymatic cleavage by enterokinase between the coding sequences for the two polypeptides, are inserted into vectors containing an appropriate promoter such as Lac, Tip, Tac, PI, T3, T7, SP6, SV40 etc. The above constructs may further contain transcriptional enhancers and ribosome binding sites between the promoters and the coding sequences. The resulting constructs are inserted into various plasmids.
DNAs encoding the desired proteins or peptides are inserted into 3' terminus of these vectors. The resulting expression vectors are introduced into appropriate hosts and the constructed transformants are cultured to produce the desired fusion proteins. The transformants are prepared by introducing the expression vectors producing the interleukin-2-calcitonin fusion proteins into suitable or appropriate hosts by the method of Hanahan (Hanahan.d.1985, DNA cloning, 1,109-135,1RS press). Particularly, E. coli BL21DE3 codons plus were transformed with the expression vector.
Also included in an embodiment of IL2-CT fusion proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the IL2-CT fusion protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. Variant IL2-CT fusion proteins may also be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity as the naturally occurring analog, although variants can also be selected which have modified characteristics. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for
example, M13 primer mutagenesis and LAR mutagenesis. Screening of the mutants is done using assays of IL2-CT fusion protein activities.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a
final derivative. Generally these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be tolerated in certain
circumstances. When small alterations in the characteristics of the IL2-CT fusion protein
are desired, substitutions are generally made in accordance with the following chart:
Original Residue Exemplary Substitutions
Asn Gln, His
His Asn, Gln
Ile Leu, Val
Leu lie, Val
Lys Arg, Gin, Glu
Met Leu, lIe
Phe Met, Leu, Tyr
Tyr Trp, Phe
Val ILe, Leu
The fusion proteins containing Calcitonin can be produced by culturing the transformed recombinant E. coli under suitable conditions. Cell extracts are obtained by treatment with lysozyme digestion, freezing and thawing, ultrasonication or French press, followed by methods, such as solubilization of extracts, ultra filtration, dialysis, ion exchange chromatography, gel filtration, electrophoresis and affinity chromatography. Calcitonin is isolated by enterokinase digestion.
METHODS OF TREATMENT
The fusion protein of the present invention is suitable for treating cancers such as breast cancers, pancreatic cancers. Alternatively, the fusion protein can be cleaved by enzymatic digestion to give Calcitonin, which can also be used in the treatment of Calcitonin indicated conditions. During hypocalcemia calcitonins reduce elevated plasma calcium concentration to normal levels by inhibiting bone resorption. Calcitonins are therefore used to treat a variety of conditions such as Paget's disease, post menopausal osteoporosis and also to treat hypocalcemia resulting from vitamin D intoxication, neoplastic disease, thyrotoxicosis or hyperthyroidism.
Both IL-2 and Calcitonin have been indicated for the treatment of cancer. The present invention is advantageous in that it provides a compound that is suitable for use in the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumors. The types of cancer that may be treated with the present invention include but are not limited to: breast, colon, prostate, thyroid, testis, melanoma, corpus and uterus, Hodgkin's lymphoma, urinary, bladder, cervix, uteri, larynx, rectum, kidney and renal, pelvis, oral cancer, pharynx, non-Hodgkin lymphoma, leukemia, Kaposi's sarcoma, ovary, brain and ONS, myeloma, stomach, esophagus, lung and bronchus, mesothelioma, liver and pancreas.
The fusion protein can be used for the treatment of IL-2 indicated conditions. IL-2 is approved by the U.S. Food and Drug Administration (FDA) for the treatment of kidney cancer and, as of 2005, also used in the treatment of HIV and AIDS. Inhaled interleukin-2 may halt disease progression in patients with kidney cancer that has spread to the lungs. Aldesleukin, a synthetic version of interleukin-2, is used to treat cancer of the kidney and skin cancer that has spread to other parts of the body. Aldesleukin is approved by the United States Food and Drug Administration (FDA) for treatment of meta-static malignant melanoma (skin cancer that has spread to other parts of the body) and metastatic renal cell carcinoma (kidney cancer that has spread to other parts of the body). It has also been used in combination with other drugs in treatment of AIDS and
cutaneous T-cell lymphoma. The availability of recombinant human interleukin 2 (rhlL-2) has resulted in its clinical utilization both as a single agent and in combination with lymphokine-activated killer cells.
The use of interleukin-2 (IL-2) in the treatment of cancer has shown limited efficacy and dose-limiting toxicity. Combination therapy with other cytokines and/or chemotherapeutic agents has been attempted to enhance the antitumor activity and to reduce the effective therapeutic dose of IL-2. Suitable anti-proliferative drugs or cytostatic compounds to be used in combination with the agents of the invention include anti-cancer drugs. Anti-cancer drugs are well known and include: Acivicin®; Aclarubicin®; Acodazole Hydrochloride®; Acronine®; Adozelesin®; Aldesleukin®; Altretamine®; Ambomycin®; Ametantrone Acetate®; Aminoglutethimide®; Amsacrine®; Anastrozole®; Anthramycin®; Asparaginase®; Asperlin®; Azacitidine®; Azetepa®; Azotomycin®; Batimastat®; Benzodepa®; Bicalutamide®; Bisantrene Hydrochloride®; Bisnafide Dimesylate®; Bizelesin®; Bleomycin Sulfate®; Brequinar Sodium®; Bropirimine®; Busulfan®; Cactinomycin®; Calusterone®; Caracemide®; Carbetimer®; Carboplatin®; Carmustine®; Carubicin Hydrochloride®; Carzelesin®; Cedefingol®; Chlorambucil®; Cirolemycin®; Cisplatin®; Cladribine®; Crisnatol Mesylate®; Cyclophosphamide®; Cytarabine®; Dacarbazine®; Dactinomycin®; Daunorubicin Hydrochloride®; Decitabine®; Dexormaplatin®; Dezaguanine®; Dezaguanine Mesylate®; Diaziquone®; Docetaxel®; Doxorubicin®; Doxorubicin Hydrochloride®; Droloxifene®; Droloxifene Citrate®; Dromostanolone Propionate®; Duazomycin®; Edatrexate®; Eflornithine Hydrochloride®; Elsamitrucin®; Enloplatin®; Enpromate®; Epipropidine®; Epirubicin Hydrochloride®; Erbulozole®; Esorubicin Hydrochloride®; Estramustine®; Estramustine Phosphate Sodium®; Etanidazole®; Etoposide®; Etoposide Phosphate®; Etoprine®; Fadrozole Hydrochloride®; Fazarabine®; Fenretinide®; Floxuridine®; Fludarabine Phosphate®; Fluorouracil®; Flurocitabine®; Fosquidone®; Fostriecin Sodium®; Gemcitabine®; Gemcitabine Hydrochloride®; Hydroxyurea®; Idarubicin Hydrochloride®; Ifosfamide®; Ilmofosine®; Interferon AIfa-2a®; Interferon Alfa-2b®; Interferon Alfa-nl®; Interferon Alfa-n3®; Interferon Beta-I a®; Interferon Gamma-I b®; Iproplatin®; Irinotecan
Hydrochloride®; Lanreotide Acetate®; Letrozole®; Leuprolide Acetate®; Liarozole Hydrochloride®; Lometrexol Sodium®; Lomustine®; Losoxantrone Hydrochloride®; Masoprocol®; Maytansine®; Mechlorethamine Hydrochloride®; Megestrol Acetate®; Melengestrol Acetate®; Melphalan®; Menogaril®; Mercaptopurine®; Methotrexate®; Methotrexate Sodium®; Metoprine®; Meturedepa®; Mitindomide®; Mitocarcin®; Mitocromin®; Mitogillin®; Mitomalcin®; Mitomycin®; Mitosper®; Mitotane®; Mitoxantrone Hydrochloride®; Mycophenolic Acid®; Nocodazole®; Nogalamycin®; Ormaplatin®; Oxisuran®; Paclitaxel®; Pegaspargase®; Peliomycin®; Pentamustine®; Peplomycin Sulfate®; Perfosfamide®; Pipobroman®; Piposulfan®; Piroxantrone Hydrochloride®; Plicamycin®; Plomestane®; Porfimer Sodium®; Porfiromycin®; Prednimustine®; Procarbazine Hydrochloride®; Puromycin®; Puromycin Hydrochloride®; Pyrazofurin®; Riboprine®; Rogletimide®; Safingol®; Safingol Hydrochloride®; Semustine®; Simtrazene®; Sparfosate Sodium®; Sparsomycin®; Spirogermanium Hydrochloride®; Spiromustine®; Spiroplatin®; Streptonigrin®; Streptozocin®; Sulofenur®; Talisomycin®; Taxol®; Taxotere®; Tecogalan Sodium®; Tegafur®; Teloxantrone Hydrochloride®; Temoporfin®; Teniposide®; Teroxirone®; Testolactone®; Thiamiprine®; Thioguanine®; Thiotepa®; Tiazofurin®; Tirapazamine®; Topotecan Hydrochloride®; Toremifene Citrate®; Trestolone Acetate®; Triciribine Phosphate®; Trimetrexate®; Trimetrexate Glucuronate®; Triptorelin®; Tubulozole Hydrochloride®; Uracil Mustard®; Uredepa®; Vapreotide®; Verteporfin®; Vinblastine Sulfate®; Vincristine Sulfate®; Vindesine®; Vindesine Sulfate®; Vinepidine Sulfate®; Vinglycinate Sulfate®; Vinleurosine Sulfate®; Vinorelbine Tartrate®; Vinrosidine Sulfate®; Vinzolidine Sulfate®; Vorozole®; Zeniplatin®; Zinostatin®; Zorubicin Hydrochloride®.
Other anti-cancer drugs suitable for combination therapy include: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta-lactam derivatives; beta-alethine; betadamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castano spermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-I receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
Ientinan sulfate; lcptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; Jissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatain A; mafimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anti cancer compound; mycaperoxide B; rnycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paelitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porflmer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras faniesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
reteiliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; saflngol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfm; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
COMPOSITIONS AND FORMULATIONS
The fusion protein of the present invention can be incorporated into a pharmaceutical composition suitable for oral administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, absorption delaying agents and the like, compatible with pharmaceutical administration. The use of such agents or carriers is well known in the prior art. A pharmaceutical composition of the fusion protein of the present invention is formulated to be compatible with its intended route of administration.
Proteins and polypeptides have been lyophilised in order to prepare powders, which may be stored and reconstituted when required. The proteins and polypeptides are freeze dried in the presence of cryoprotectants and lyoprotectants (e.g. sugars, polyols, polymers such as polyethylene glycol, amino acids and organic salts such as sodium acetate) which are required to prevent the denaturation of the protein or polypeptide. Some proteins and polypeptides are freeze dried in the presence of cryoprotectants and lyoprotectants but these techniques have not been utilized before to prepare solid oral preparations of unstable proteins or polypeptides such as those having a disulphide bridge. In particular these techniques have not before been utilised to provide a stable oral formulation of calcitonins.
The present invention is suited to the development of prodrugs—a class of drugs whose pharmacologically active entity is released when the drug is metabolized within the body. Prodrugs designed for oral administration are based on an in-depth knowledge of how the digestive system works. Absorption of some oral medication is compromised by the chemical process called protein hydrolysis taking place within the gastrointestinal tract that rapidly breaks down food and drug compounds into elementary proteins, amino acids and other nutrients. Some medications, particularly those with a large and complex molecular structure, are particularly vulnerable to this process as with Calcitonin., where less than 1 percent of Calcitonin ever makes it to the patient's bloodstream About 60 percent bioavailability is desirable in new drug compounds, but 10 to 20 percent is acceptable when there are no alternatives. Using a series of biochemical procedures, one amino acid cluster is removed from the peptide molecule and replaced with a specific prodrug compound designed to resist the destructive metabolic processes within the GI tract. Once in the intestine, peptide molecules—carrying the prodrug "hidden" within them—pass easily into cells lining the wall of the intestine. Once inside these cells, the peptide prodrug reacts chemically with enzymes present in the cell fluid to produce the drug's active ingredient, which then diffuses through the outer intestinal cell wall into blood vessels on the other side. These prodrug techniques have the potential to improve the effectiveness of many hard-to-absorb oral medications currently used to treat
cardiovascular diseases, osteoporosis and other illnesses. Improving absorption also may help reduce side effects compounds that remain in the digestive system.
In order to formulate proteins and polypeptides into pharmaceutical preparations the factors described above must be taken into consideration. Thus proteins and peptides often have more complex formulation requirements than chemical pharmaceuticals. This is further complicated by the fact that many stages in the processing of pharmaceutical formulations introduce further stresses on the proteins and polypeptides, which destabilize them. For example, processes such as heating, shaking, freeze thawing and processes in which the proteins or polypeptides are exposed to hydrophobic surfaces or to moisture may induce aggregation of the protein or polypeptide. Aggregation may also occur during the storage of the formulation, particularly if it is exposed to moisture.
Various excipients such as albumin, amino acids, sugars, chelating agents, cyclodextrins and polyhydric alcohols, have been added to proteins and polypeptide pharmaceutical formulations in order to increase their stability. These have been of varying success depending on the protein or polypeptide concerned. The excipients stabilize the proteins and polypeptides in different ways, not all of which are fully understood, for example, albumin is added to prevent surface adsorption of pharmaceuticals by preferentially adsorbing to surfaces, whilst amino acids are added to reduce surface adsorption, to inhibit aggregation or to reduce heat degradation. Sugars are added to provide stability during processes such as heating and lyophilisation.
The peptides of the invention can be compounded with a pharmaceutically acceptable, monomeric, soluble carrier for ease of manufacture and/or administration. Examples of carriers include polyalcohols such as mannitol and sorbitol, sugars such as glucose and lactose, surfactants, organic solvents, and polysaccharides. Thus in another aspect the composition of the invention further comprises a pharmaceutically acceptable, soluble, monomeric carrier, e. g. , mannitol, sorbitol, lactose, and the like, said monomeric carrier preferably being present in an amount of up to 30 percent by weight of the dry weight of said composition.
Sustained-release formulations have been developed to deliver peptides over prolonged periods of time without the need for repeated administrations. Solid polymeric microcapsules and matrixes, for example, utilizing biodegradable polylactic polymers, have been developed. See e. g. , Hutchinson, U. S. Pat. No. 4,767, 628 and Kent, et al. , U. S. Pat. No. 4, 675, 189. Hydrogels have also been used as sustained-release formulations for peptides. These hydrogels comprise polymers such as poly-N-isopropyl acrylamide (NIPA), cellulose ether, hyaluronic acid, lecithin, and agarose to control the delivery. See, e. g., PCT Applications WO 94/08623.
The IL2-CT fusion protein compositions described herein may be used in the preparation of a formulation, such as a pharmaceutical formulation, by combining the IL2-CT fusion protein composition(s) with a pharmaceutically acceptable carrier, excipients, stabilizing agents or other agent, which are known in the art, for use in the methods of treatment, methods of administration, and dosage regimes described herein. To increase stability by increasing the negative zeta potential of nanoparticles, certain negatively charged components may be added. Such negatively charged components include, but are not limited to bile salts, bile acids, glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid, dehydrocholic acid, and others; phospholipids including lecithin (egg yolk) based phospholipids which include the following phosphatidylcholines: palmitoyloleoylphosphatidylcholine,
stearoyloleoylphosphatidylcholine, stearoylarachidoylphosphatidylcholine, and
dipalmitoylphosphatidylcholine. Other phospholipids including L-a-
dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and other related compounds. Negatively charged surfactants or emulsifiers are also suitable as additives, e.g., sodium cholesteryl sulfate and the like.
In some embodiments, the IL2-CT fusion protein composition is suitable for administration to a human. There are a wide variety of suitable formulations of the
inventive composition (see, e.g., U.S. Pat. Nos. 5,916,596 and 6,096,331, which are hereby incorporated by reference in their entireties). The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration can comprise (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, (d) suitable emulsions, and (e) powders. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
The formulations or compositions that contain the fusion protein of the present invention can have a therapeutic concentration though the amount varies with the dosage form of the medicament.
Administration depends on the body weight of the patients, the seriousness of the conditions and the doctor's opinion. The dose of the composition will depend on the type of disease (such as cancer) to be treated, the severity and course of the disease, the individual's clinical history, and the discretion of the attending physician. Suitable dosages of the IL2-CT fusion protein in a pharmaceutical composition include, but is not limited to, about any of 20 mg/m2 of body surface, 30 mg/m2, 40 mg/m2, 50 mg/m2, 70 mg/m2, 90 mg/m2, 100 mg/m2, 125 mg/m2, 150 mg/m2, 175 mg/m2, 180 mg/m2, 200 mg/m2, 210 mg/m2, 220 mg/m2, 240 mg/m2, 250 mg/m2, 280 mg/m2, and 300 mg/m2.
The dose can be administered once or several times daily according to the severity of the conditions.
The compositions or formulations of the fusion protein of the present invention can be co-administered with one or more therapeutic agents.
In some embodiments, the composition is administered at least about any of once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily. Other exemplary dosing frequencies include, but are not limited to, weekly, two Out of three weeks; weekly, three out of four weeks; and weekly, four out of five weeks. In some embodiments, the composition is administered (with or without breaks in administration cycles) for at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more month(s). In some embodiments, the composition is administered via any of intravenous, intraperitoneal, or inhalational routes.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: CLONING OF IL-2 IN CLONING VECTORS.
The process comprises
a. Isolation of RNA from cultured Jurkat cells
b. Construction of cDNA from RNA.
c. The PCR amplification of cDNA is done to give IL-2 with signal sequence.
d. Ligation of IL-2 with signal sequence with pGEMT vectors to yield IL2pGEMT-l
e. Transformation of the ligated IL2pGEMT-l mix in XL-1 competent cells yields
clones of plasmids.
f. PCR amplification of one of the clones of pGEMTIL-2 plasmids to yield IL-2 without
signal sequence with appropriate primers.
g. Ligation of IL-2 without signal sequence with pGEMT vectors to yield clones of
plasmids to yield IL2pGEMT-2
h. Transformations of ligated IL2pGEMT-2 mix in XL-1 competent cells yields clones
of plasmids. i. Ligation of IL-2 from one of the clones of IL2pGEMT-2 plasmids with pET 24 A
vectors to yield IL2 pET j. Transformation of ligated IL2pET in ToplOF' cells k. Site directed mutagenesis to yield des alanyl IL-2 of IL2 pET 1. PCR amplification to yield IL2 without stop codon.
a) Isolation of RNA from cultured Jurkat cells
Jurkat cells were grown till 60-70% confluency and they were pelleted. After aspiration of the medium the pellet was lysed with RLT buffer and homogenized. The lysate was mixed with 70% ethanol and loaded on Rneasy column (Qiagen) where it was washed with buffers like RW1 and RPE and subsequently RNA was eluted in RNAse free water.
b) Construction of cDNA from RNA
5 ul of the RNA eluted above was then mixed with 4 ul of dNTP and 2 ul of oligo dT. After making up the volume with 5 ul sterile water, it was mixed and centrifuged. Further heated for 3 minutes at 70°C and replaced on ice. Subsequent addition of 2 ul of lOx RT buffer, lul of M-MLV RT and lul of RNase inhibitor to the mixture, it was incubated at 35-50°C for one hour. After the RT reaction, it was stored at a temperature of-20°C till further use.
c) PCR amplification of cDNA to yield IL-2 without signal sequence (figure 1)
The following primers were used to amplify IL-2 with signal sequence from the c-DNA, TPG195- forward primer for amplifying IL-2 from jurkat cells with signal sequence
5'd-(CTCTCTTTAATCACTACTCA)-3' [SEQ ID No.5]
TPG196-reverse primer for amplifying IL-2 from jurkat cells with signal sequence
To 5 µl of the c-DNA, 18 µl of 1 Ox pfu buffer, 4 µl of d NTPS and 10 ul of each primer was added and the volume was made up to 180 µl. An enzyme mix containing 2 µl of 10 x pfu buffer and 1.6 pi of native pfu in a total volume of 20 µl was made separately and added to the first tube. The PCR conditions followed were 95°C for 30 sec, 58°C for 30sec, 72°C for lmin, 2°C for 7mins, and kept at 4°C till further use. The reaction was cycled 20 times. The PCR product was loaded on a 1.5% agarose gel .It showed a very good amplification of expected size of about 500 bp.
d) Ligation of IL-2 with signal sequence with pGEMT vectors to yield
The IL-2 fragment was gel eluted using the Qiagen gel extraction kit and ligated to pGEMT vector in a volume of 10 µl. The constituents of the ligation mix was 5 µl of 2x rapid ligation buffer,3 ul of PCR product, 1 µl of pGEMT vector and 1 pi of T4 DNA ligase. The ligation was carried out at 4°C for 16-18 hours.
e) Transformation of of IL2pGEMT-l into XL-1 competent cells (Figure 2)
IL-2pGEMT-l plasmid ligation mix was transformed into competent XL-1 cells following standard transformation protocols ( Maniatis et.al) and the transformants were plated on LB-lX-gal-AMP plates. The plates were incubated at 37°C overnight. Plenty of white colonies were seen on the plate, out of which 10 were inoculated into LB broth for plasmid preparation. Plasmids were made from these cultures by Qiagen method and the plasmids were digested with EcoR-1. All of them released the right sized fragments.
f) PCR amplification of Clones of plasmid to yield IL-2 without signal sequence
PCR amplification was done using one of the plasmids generated above as the template and the following primers;
TPG239-Forward primer for cloning IL-2 with Nde-1 at 5' end 5'd-(CATATGGCACCTACTTCAAGTTCTAC)-3' [SEQ ID No.6]
TPG 240-Reverse primer for cloning IL-2 with BamHl at 3' end
5'd-(GGATCCTTATCAAGTCAGTGTTGAGA)-3' [SEQ ID No.7]
To 5 ul of the template, 18 µl of lOx pfu buffer, 4 µl of d NTP'S and 10 ul of each primer was added and the volume was made up to 180 ul. An enzyme mix containing 2 ul of lOx PFU buffer and 1.6 ul of native pfu in a total volume of 20 µl was made separately and added to the first tube. The PCR conditions followed were 95°C for 30 sec, 58°C for 30sec, 72°C for 1 min, 2°C for 7mins, and kept at 4 °C till further use. The reaction was cycled 20 times. The PCR product was loaded on a 1.5% agarose gel . It showed a very good amplification of expected size.
g) Ligation of IL-2 without signal sequence with pGEMT vectors to yield IL2pGEMT-2
The IL-2 fragment was gel eluted using the Qiagen gel extraction kit and ligated to pGEMT vector in a volume of 10 ul. The constituents of the ligation mix was 5 ul of 2x rapid ligation buffer,3 ul of PCR product, 1 µl of pGEMT vector and 1 ul of T4 DNA ligase. The ligation was carried out at 4°C for 16-18 hours.
h) Transformation of IL-2pGEMT-2 into XL-1 competent cells
IL2pGEMT-2 plasmid ligation mix was transformed into competent XL-1 cells following standard transformation protocols. The transformants were plated onto LB-X-GAL/IPTG plates. The plates were incubated at 37°C. Eight colonies were picked up for plasmid preparation and for further analysis. They were inoculated into LB broth and plasmids made by Qiagen method. The plasmids were digested with Nde and Bam H-l six of them released the IL-2 fragment, of the expected size. The six DNAs were sent for sequencing. The sequences matched perfectly to the known IL-2 sequence. The Nde-Bam H-l fragment was eluted from the gel and ligated to the expression vector, pET 24a, digested with Nde-1 and Bam H-1.
i) Ligation of IL-2 from IL2pGEMT-2 with pET vectors
5 ul of the insert, 1 ul of the vector, 1 ul of the ligase buffer and 1 ul of the T4 DNA
ligase was mixed together in a tube and incubated at 16°C overnight.
j) Transformation of IL2pET 24A into Top 10 F'-
IL2-pET24A plasmid ligation mix was transformed into competent XL-1 cells following standard transformation protocols. The transformants were plated onto LB-Kanamycin plates. The plates were incubated at 37°C. Eight colonies were picked up for plasmid preparation and further analysis. They were inoculated into LB broth and plasmids made by Qiagen method. The plasmids were digested with Nde-1 and Bam H-l six of them released the IL-2 fragment, of the expected size. The six DNAs were sent for sequencing .The sequences matched perfectly to the known IL-2 sequence. (Figure 3)
k) Site directed mutagenesis of IL2pET 24A to get des-alanyl IL-2
A site directed mutagenesis of this plasmid was done to change 125 Cys to 125 Ser. The following primers were used to bring about the change. TPG273-Forward primer with Nde-1 and without first alanine CATATGCCTACTTCAAGTTCTACAAA [SEQ ID No.8]
TPG-274-reverse primer for amplification of IL-2 with BamHl site and Cys-125 changed to Ser-125 for creating IL-2 , PROLEUKIN
PCR amplification was done with both Taq polymerase and pfu. The PCR conditions followed were 94°C for 2 mins, 94°C for 30 sees, 58°C for 30 sees, 72°C for 1 min, 72°C for 7 mins and kept at 4°C till further use. The PCR product was resolved on a 1.2% agarose gel and eluted, using gel extraction kit from Qiagen. The gel fragment was ligated to pGEMT vector and transformed into competent Top 10 F' cells and cells were plated onto LB-X-GAL IPTG plates and they were incubated at 37°C overnight. A few colonies were inoculated into LB broth and plasmids made from them and the plasmids were digested with Nde-Bam. The Nde-Bam fragment was ligated to pET vector digested with Nde and Bam-Hl and the ligation mix was transformed into competent Top 10F' cells. The cells were plated on LB+ kanamycin medium and incubated at 37°C overnight. A few colonies were inoculated in LB+kanamycin broth and incubated overnight. Plasmids were made from them and the plasmids analysed by restriction digestion with Nde and bam-hl.colonies releasing the IL-/EK-CT were taken forward for expression
purpose. Two clones were sent for sequencing and the sequence data showed perfect alignment with Nde-Bam HI sites intact.
1) PCR to generate IL-2 without stop codon.
A PCR was done with the above generated clone as template and primers TPG 273 and
5'-d(CATATGCCTACTTCAAGTTCTACAAA)-3' [SEQ ID No.9]
To 5 ul of the c-DNA, 18 µl of lOx pfu buffer, 4 ul of d NTP'S and 10 µl of each primer was added and the volume was made up to 180 ul. An enzyme mix containing 2 ul of 1 Ox pfu buffer and 1.6ul of native pfu in a total volume of 20 ul was made separately and added to the first tube. The PCR conditions followed were 95°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 2°C for 7min, and kept at 4°C till further use. The reaction was cycled 20 times. (Figure 4)
EXAMPLE 2: CLONING OF CALCITONIN WITH VECTORS
A) Designing of oligos for synthesis of human Calcitonin;
The following oligos were designed for the synthesis of human Calcitonin for expression
in either E. coli or Pichia.
5'd-(TGTGGTAACTTGTCTACTTGTATGTTGGGTACTTACACTCAAGAT TTTAACAAGTTTCAT)-3' [SEQ ID No.10] Primer-2- [SEQ ID No. 11]
5'd-(ACTTTTCCACAAACTGCTATTGGTGTTGGTGCTCCATAAGAATTC)-3' Primer-3- [SEQ ID No. 12]
Primer-4- [SEQ ID No. 13]
B ) Annealing of Calcitonin oligos;
The oligos were suspended to lOOµM in Tris- EDTA- NaCl (TEN), and 15 µl of sense and antisense oligos were mixed in 200 µl PCR tube. The tube was incubated for 2-10 minutes at 90-100°C in thermal cycler and then the tubes were transferred to a beaker containing boiling water. The contents were stirred until the temperature of the water was equilibrated to 25-370C. The annealed product was used as a template for PCR amplification.
C) PCR and cloning of calcitonin into cloning vector 0
A gradient PCR was done at various temperatures with the annealed mixture as the
template and the primers used were TPG 113 and TPG 114.
TPG-113- 5M-(TGTGGTAACTTGTCTACTTG)-3' [SEQ ID No. 14]
5'd-(TPG114-GAATTCTTATGGAGCACCAA)-3' [SEQ ID No.15]
The PCR mix comprised of 18µl of lOx pfu buffer, 4 µl of dNTP's lOµl of TPG 113 and TPG 114 primers, 0.5ul of template and 137.5µl of water. The enzyme mix comprised of 2 µl of lOx µl, 1.6µl of pfu and 16.4 µl of water, which was added to the main PCR mix.
The PCR conditions followed were 94°C for 5 mins, 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, 72°C for 1 min and finally kept at 4°C till further requirements. The reaction was cycled for 29 times. The PCR product was loaded on a 2% agarose gel (Figure 5). A Re- PCR was done at 55°C for 30 sec. The PCR product was loaded one a 2% agarose gel andband eluted using gel extraction kit of Qiagen. The band was eluted in 10 ul of sterile water. The fragment was ligated into pUC 19 vector and the ligation mix was transformed into Top 10F' competent cells. The transformants were plated onto LB-X-GAL-IPTG amp; plates and incubated at 37°C. ( Figure 6)
D) Screening of positive clones and their analysis
A few white colonies were grown in LB broth, and plasmids were made out of them. The plasmids were digested with Ecol and BamH-1 enzymes, and the clones, which released the lOObp calcitonin fragment, and were taken forward for study.
£) Sequencing of calcitonin
The pUC clone containing calcitonin was sent for sequencing. Sequencing was done by Sanger's dideoxy method and the sequence was found to perfect, as it aligned with our original sequence.
F) PCR amplification to generate Calcitonin with enterokinase site at 5' end and glycine at 3'end.
PCR amplification was done using the following primers
TPG 228-forward primer for amplifying calcitonin with enterokinase cleavage site at the
N terminus of calcitonin.
5'd- (GACGACGACGACAAGTGTGGTAACTTG)-3' [SEQ ID No.16]
TPG-236-reverse primer for calcitonin with bamhl and glycine at 3' end before stop
codon for amidation purpose.
The PCR conditions followed were at 94°C for 5 rnins, 94°C for 30 sec, 55°C for 30sec,
72°C for 30sec, 72°C for 1 minute and kept at 4°C till further requirement. The PCR
product was loaded on a 2% agarose gel and the band eluted using gel extraction kit of
Qiagen. The band was eluted in 10 µl of sterile water. The fragment was ligated into
pGEMT vector and the ligation mix was transformed into top 10F' competent cells. The
transformants were plated onto LB-X-GAL-IPTG amp plates and incubated at 37°C.(
Lanes 3,4 of figure 4)
EXAMPLE 3: FUSION OF CLONED IL-2 WITH CLONED CALCITONIN.
Ligation of equimolar concentrations IL-2 without stop codon of step J of example 1 and Calcitonin with enterokinase at 5' end of step F of example 2 was done together by T4 DNA ligase in the presence of ligase buffer and incubated overnight at 16°C.
EXAMPLE 4: PCR TO AMPLIFY THE FUSED PRODUCTS
The above generated ligation mix was used as the template and the following primers were used to amplify IL-2/EK-CT.
The PCR product was resolved on a 1.2% agarose gel and eluted, using gel extraction kit from Qiagen. (Figure 7)
EXAMPLE 5: LIGATION OF FUSED IL-2 - CALCITONIN INTO VECTORS.
The gel fragment was ligated to pGEMTvector and transformed into competent Top 10 cells and cells were plated onto LB-X-GAL IPTG plates and they were incubated at 37°C overnight. (Figure 8)
Screening of the colonies and subcloning of IL-2-Calcitonin fusion protein into pET 24 A vector
A few colonies were inoculated into LB broth and plasmids made from them and the
plasmids were digested with Nde-Bam (Figure 9). The Nde-Bam fragment was ligated to pET24A vector digested with Nde and BamHl and the ligation mix was transformed into competent top 10 F' cells. The cells were plated on LB+ kanamycin medium and incubated at 37°C overnight. Two clones were sent for sequencing and the sequence data showed perfect alignment with Nde-Bam Hi sites intact. ( Figure 10)
EXAMPLE 5: TRANSFORMATION OF LIGATED FUSED IL-2- CALCITONIN INTO EXPRESSION VECTORS
3 ul of the plasmid pep was transformed into competent BL21DE3 by standard transformation protocols and the transformants were plated on LB+kanamycin plates. Plenty of colonies could be seen on the plate. A couple of the positive clones were taken for expression studies.
EXAMPLE 6: STUDIES OF EXPRESSION.
One of the positive clones was inoculated into 10 ml of LB+kan broth and grown to an o.d of 0.6 The culture was induced with 1 Mm IPTG and the flask kept back at 37 deg shaker for 3 hours. Samples were collected post induction at 3hours and one sample before induction. The samples were normalized to and od of 1.0 for comparative purpose. The samples were spun down. The cells were processed by adding lx SDS loading buffer ,boiled for 7 mins ,and loaded on two 15 % SDS-PAGE. The gel was run at 150constant volts and one of the gels stained with commasie blue and another gel was blotted onto nitrocellulose paper for probing with antibodies to verify the proteins authenticity. The comassie stained gel showed a clear and distinct band of the required size which gave a
sharp signal with anti-IL-2 antibody on one gel and anti-calcitonin antibody on another gel proving that the band we detect is indeed IL-2-CT fusion.
EXAMPLE 7: CULTURING OF FUSED PROTEIN IN FERMENTOR.
50ml of filter sterilized CDM ( chemically defined medium) with 30mg/ml of Kanamycin and lOmg/ml of Thiamine was inoculated with 50µl E. coli frozen stock and was kept in INNOVA shaker at 200 rpm and 37°C for 10-12 hours. Seed Optical density was 1.0. Fermentation parameters were set at agitation of 30-960 and DOT at 30% with a temperature of 30-40°C at pH 6-7.0. The fermentation (about 4 Litre batch size) was inoculated with 40 ml of inoculum a nd at 8-10 OD batch ws induced with 1 mM IPTG. After induction the batch was continued for 6 hours for cell growth and protein The culture was harvested in sterile centrifuge tubes and centrifuged. Supernatent was discarded and pellet was used for downstream processing,
EXAMPLE 8: PURIFICATION OF THE FUSED IL-2- CALCITONIN PROTEIN.
A positive clone, was grown in 50 ml LB-broth containing kanamycin to log phase, and this culture was inoculated into 2 liters of LB-broth containing kanamycin, grown to and O.D. of 0.6 and then induced with IPTG(1 mM) and grown to 3 hours. The cells were harvested in cold and the cell pellet collected.
INCLUSION BODIES PREPARATION (figureII)
1. cell pellet was weighed and suspended in a volume of lysis buffer that is twice that of the pellet weight and sonicated by giving 12 pulses.
2. The suspension is diluted to ten times the pellet weight with lysis buffer and 6 more pulses are given and cell lysis is monitored by absorbance at 600 nm till there is no change in OD.
3. After lysis the suspension is centrifuged and the pellet is subjected to a series of washes with wash buffer containing 1% tritonX 100.
4. Finally the pellet is washed with tris uffer to remove tritonX 100.
5. The final pellet is the inclusion bodies.
SOLUBLISATION OF INCLUSION BODIES (figure 12)
1. The inclusion bodies are solubilised in 8M urea in 1:40 ratio (weight/volume) and the pH is adjusted to 12 with 1M NaOH.for two hours.
2. After solubilization the sample is centrifuged to remove unsolubilised aggregates.
3. The supernatent is collected.
REFOLDING (figure 13)
1. The solubilised material is subjected to refolding by maintaining a final concentration of 0.5mg/ml.
2. The refolding is done at room temperature for 16 hours.
3. After refolding the sample is filtered and is either diluted or dialysed to make the conductivity conducible for loading onto column for chromatography.
FIRST COLUMN CHROMATOGRAPHY
1. The diluted or dialysed sample is loaded onto DEAE sepharose pre equilibrated with 50mMtrispH8.5.
2. The column is washed with 50mM tris pH 8.5.
3. The protein is eluted with 50mM tris pH8.5 containing 80mM NaCl.
EXAMPLE 9: CLEAVAGE OF FUSED PROTEIN WITH ENTEROKINASE
Cleavage of the fusion peptide was monitored by SDS polyacrylamide gel electrophoresis if the recombinant protein is small enough to see a size shift following its removal. Enterokinase digestion yielded the wild type protein with no additional amino acids at the amino terminus. Enterokinase digestion was carried out overnight at 4°C with an enterokinase concentration of 5 units/mg of synthetase. Under these conditions the cleavage efficiency was 100%, with minimal ( Following enterokinase digestion, the His-tag was then separated from the free enzyme with a second Ni-NTA affinity chromatography. Purified fusion NADP-ME protein was then incubated with enterokinase (1:100) in buffer B at 10°C for 2 hours to remove the NH2 terminus codified by the expression vector.
The products of digestion were separated with size exclusion chromatography (SEK) on Superdex 200. The protein sample was about 98% pure according to Coomassie blue
stained SDS-PAGE and was used for crystallization trails. The protein was concentrated till 15mg/ml, in 50mM TrisCl (pH 8.0), 300mM NaCl and lOmM of fi-mercaptoethanol. The protein was crystallized in 0.4M ammonium sulfate and 35% of PEG 8K at pH 6.5 in cacodylate buffer. The crystals looked as small thin plates. The products of digestion were separated with size exclusion chromatography (SEK) on Superdex 200. The protein sample was about 98% pure according Coomassie blue stained SDS-PAGE and was used for crystallization trails. The protein was concentrated till 15mg/ml, in 50mM TrisCl (pH 8.0), 300mM NaCl and lOrnM of β-mercaptoethanol. The protein was crystallized in 0.4M ammonium sulfate and 35% of PEG 8K at pH 6.5 in cacodylate buffer. The crystals looked as small thin plates.
The protein was further purified using an affinity Affi-Gel blue column (Bio-Rad) followed by a second nickel-NTA column. The purified enzyme was stored at 80°C in buffer B (with 50% glycerol) for further studies. Protein concentration was determined by the method of Sedmak and Grossberg et. Anal Biochem 1977 May 79 (1-2) 544-552 using bovine serum albumin as standard.
EXAMPLE 10: BIO ASS AY/ TOXICITY STUDIES
The human breast cancer cell line T47D expresses Calcitonin (CT) receptors that are coupled to adenylate cyclase and which reveal a dose dependent cyclic AMP response to CT. This assay is used for CT preparations as well as IL2 or CT preparations. (Grover et.al , Bone Miner 1992, Apr 11(1) 65-74) T47 D invitro bioassays is more sensitive, superior in precision and accuracy and comparable in specificity to the rat hypocalcemia. The Calcitonin can be used to inhibit invasion of breast cancer by using MOA -MB 231 cell line. An encapsulation of CT with different materials is also studied for transport mechanism using CACO-2 cell lines. ( Shibu et al, Cancer Res. 2005, 65: 8519-8529)
Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, are within the scope of the invention.
A recombinant fusion protein comprising at least one interleukin-2 sequence or variant thereof and at least one calcitonin sequence or variant thereof.
The recombinant fusion protein of claim 1, wherein the calcitonin is located at the N-terminus of said recombinant fusion protein.
The recombinant fusion protein of claim 1, wherein the calcitonin is located at the C-terminus of said recombinant fusion protein.
The recombinant fusion protein of claim 1, wherein the recombinant fusion protein retains at least part of a therapeutic activity of unfused interleukin-2.
The recombinant fusion protein of claim 1, wherein the recombinant fusion protein retains at least part of a therapeutic activity of unfused calcitonin.
The recombinant fusion protein of claim 1, wherein the recombinant fusion protein further comprises a linker peptide between each interleukin-2 sequence and calcitonin sequence, and further wherein the linker peptide is cleavable by a protease.
The recombinant fusion protein of claim 6, wherein the linker peptide comprises the sequence Asp4-Lys (D4K).
The recombinant fusion protein according to any of claims 6 or 7, wherein the protease is enterokinase.
The recombinant fusion protein of claim 1, comprising the amino acid sequence: MPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRW1TFCQSIISTLTCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPG.
The recombinant fusion protein according to any of claims 6, 7 or 8, comprising the
amino acid sequence:
The recombinant fusion protein of claim 1, wherein said recombinant fusion protein comprises an N-terminal Methionine.
The recombinant fusion protein of claim 1, wherein said recombinant fusion protein further comprises a secretion signal sequence.
The recombinant fusion protein of claim 12, wherein said secretion signal sequence is the natural leader sequence of interleukin.
The recombinant fusion protein of claim 1 or 6, wherein said interleukin is fused to the N-terminal end of calcitonin or calcitonin variant.
The recombinant fusion protein of claim 1 or 6, wherein said interleukin is fused to the C-terminal end of calcitonin or calcitonin variant.
The recombinant fusion protein of claim 1, wherein said recombinant fusion protein is expressed by a prokaryotic cell.
The recombinant fusion protein of claim 16, wherein said recombinant fusion protein is expressed by a bacteria.
The recombinant fusion protein of claim 1, wherein said recombinant fusion protein is expressed by a eukaryotic cell.
The recombinant fusion protein of claim 18, wherein said recombinant fusion protein is expressed by an animal cell.
The recombinant fusion protein of claim 19, wherein said animal cell is a CHO cell.
The recombinant fusion protein of claim 19, wherein said animal cell is a COS cell.
The recombinant fusion protein of claim 1, wherein said recombinant fusion protein is expressed by a yeast.
The recombinant fusion protein of claim 22, wherein said yeast is Saccharomyces.
The recombinant fusion protein of claim 18, wherein said recombinant fusion protein is expressed by a fungi.
A nucleic acid molecule comprising a IL2-CT polynucleotide sequence encoding the recombinant fusion protein according to any one of claims 1 through 15.
A vector comprising the nucleic acid molecule of claim 25 and a heterologous polynucleotide sequence.
The nucleic acid molecule of claim 25, comprising a sequence encoding a pharmaceutically effective variant of at least one of IL-2 and calcitonin.
The nucleic acid molecule of claim 26, wherein said heterologous polynucleotide comprises a promoter sequence operably linked to the IL2-CT polynucleotide sequence.
The nucleic acid molecule of claim 28, wherein said promoter sequence is any one selected from the group of: a hybrid promoter; a constitutive promoter; a regulatable promoter; a yeast phosphoglycerate kinase (PGK) promoter; a yeast glyceraldehyde-3-phosphate dehydrogenase (GDP) promoter; yeast lactase (LAC4) promoter; a yeast enolase (ENO) promoter; a yeast alcohol dehydrogenase (ADH) promoter; a yeast acid phosphatase (PH05) promoter; a lambda bacteriophage PL promoter; a lambda bacteriophage PR promoter; a Ptrp promoter; and a Plac promoter.
The nucleic acid molecule of claim 26, wherein said heterologous polynucleotide further comprises a selectable marker.
The nucleic acid molecule of claim 28, wherein said heterologous polynucleotide further comprises one or more of an enhancer sequence, and a ribosome binding site sequence operably linked to the promoter sequence and the IL2-CT polynucleotide sequence.
The nucleic acid molecule of claim 26, wherein said heterologous polynucleotide comprises a region for termination of transcription.
An isolated host cell comprising the nucleic acid molecule of claim 25.
An isolated host cell comprising the nucleic acid molecule of claim 26.
A method for producing a recombinant fusion protein, comprising:
culturing the isolated host cell of claims 33 or 34 under conditions suitable to produce the
recombinant fusion protein encoded by said polynucleotide; and
recovering said recombinant fusion protein.
The method of claim 35, further comprising:
contacting the recombinant fusion protein with a protease suitable for cleavage at the
linker peptide between IL-2 and calcitonin.
A recombinant fusion protein produced by the method of claim 35.
A recombinant fusion protein produced by the method of claim 36.
A therapeutically effective formulation comprising one or more recombinant fusion proteins of claim 1, and a pharmaceutical excipient.
The therapeutically effective formulation of claim 39, suitable for oral administration.
A calcitonin prodrug, comprising the recombinant fusion proteins of claims 6 or 10.
A composition comprising one or more recombinant fusion proteins of claims 1 through 15.
. A recombinant fusion protein and its methods as claimed above exemplified herein substantially in the examples and figures.
|Indian Patent Application Number||973/MUM/2006|
|PG Journal Number||29/2011|
|Date of Filing||21-Jun-2006|
|Name of Patentee||RELIANCE LIFE SCIENCES PRIVATE LIMITED|
|Applicant Address||CHITRAKOOT, 2nd FLOOR, SHREE RAM MILLS COMPOUND, GANPATRAO KADAM MARG, WORLI, MUMBAI 400013,|
|PCT International Classification Number||C071K14/435|
|PCT International Application Number||N/A|
|PCT International Filing date|