WO2015188110A1 - Reversible pegylation for screeing of therapeutic proteins in vivo - Google Patents
Reversible pegylation for screeing of therapeutic proteins in vivo Download PDFInfo
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- WO2015188110A1 WO2015188110A1 PCT/US2015/034504 US2015034504W WO2015188110A1 WO 2015188110 A1 WO2015188110 A1 WO 2015188110A1 US 2015034504 W US2015034504 W US 2015034504W WO 2015188110 A1 WO2015188110 A1 WO 2015188110A1
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- 0 *=C(CCCS)NCCCCC(C(O)=O)N(CC(O)=O)CC(O)=O Chemical compound *=C(CCCS)NCCCCC(C(O)=O)N(CC(O)=O)CC(O)=O 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
Definitions
- This invention is based, in part, on the use of the complementary interaction of a His- tag and a Ni 2+ complex of nitrilotriacetic acid (NT A) for quick therapeutic protein screening in vivo. Particularly, this specific and strong interactive pair is used to improve potency of therapeutic proteins in vivo after administration, e.g., systemic administration.
- NT A nitrilotriacetic acid
- Described herein is the PEGylation of proteins through complementary interactions between an oligohistidine tag (His-tag) and a Ni 2+ complex of nitrilotriacetic acid (NT A) to improve the half-life of therapeutic proteins in the blood following administration, e.g., systemic administration, in vivo.
- oligohistidine tag His-tag
- NT A Ni 2+ complex of nitrilotriacetic acid
- animal models that show that this site- specific modification improves the efficacy of a modified model protein.
- the fast modification is used to screen therapeutic proteins before applying time- consuming, expensive half-life extension technologies, e.g., site-specific PEGylation.
- a poly-histidine tag (His-tag) to the peptide, mixing the His-tagged polypeptide with a complex comprising a metal, nitrilotriacetic acid (NT A) and PEG (poly (ethylene glycol)) or analogue thereof, thereby producing a His-tagged peptide/ Ni-NTA-PEG composition and improving in vitro and in vivo pharmacokinetic parameters.
- NT A nitrilotriacetic acid
- PEG poly (ethylene glycol)
- the method further comprises administering the composition to a subject, e.g., a human subject.
- the His-tag comprises six or eight histidine amino acids.
- the His-tag is located at the N terminus.
- the His-tag is located at the C terminus.
- compositions of the invention are administered intravenously or systemically.
- Other modes of administration include oral, rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes.
- parenteral includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations.
- Compositions comprising a composition of the invention can be added to a physiological fluid, such as blood.
- Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
- Parenteral modalities subcutaneous or intravenous
- Inhaled therapy may be most appropriate for pulmonary vascular diseases (e.g., pulmonary hypertension).
- compositions and methods described herein improve in vitro and in vivo pharmacokinetic and pharmacodynamics properties.
- the Ni-NTA-PEG analogue/His -tagged protein results in a prolonged half-life, increased bioavailability, decreased clearance, and reduced aggregation of the His-tagged protein.
- the improvement in efficacy comprises an increase in stability of the peptide, polypeptide, or protein in solution by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
- compositions and methods described herein increase the half-life of the peptide, polypeptide, or protein by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
- the compositions and methods described herein increase the bioavailability of the peptide, polypeptide, or protein by at least 1.5 fold, e.g., 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold.
- compositions and methods described herein increase the aqueous solubility of the peptide, polypeptide, or protein by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
- the compositions and methods described herein are utilized to retain at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the bioactivity of the peptide, polypeptide, or protein.
- the His-tagged peptide/ Ni-NTA-PEG compositions are produced within about 30 minutes, e.g., within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, or within about 12 hours.
- NTA-PEG analogues comprise a structure of the following Formula (I):
- NTA represents COO " (nitrilotriacetic acid group);
- Ri is optionally substituted alkyl, optionally substituted alkoxy, or hydroxyl
- i 0 or a positive integer
- n is a positive integer
- M is a metal ion.
- i 1 or 2.
- M 1 or 2.
- ay have a molecular weight ranging from about 1,000
- n may have a linear or branched shape as being bound to at least one or more of NTA.
- m is 1 or 2.
- M is Ca 2+ , Co 2+ , Cu 2+ , Ni 2+ , or Zn 2+ .
- L may suitably conjugate with or coupled to at least one or more of NTA and amide which is further coupled to the optionally substituted ethylene oxide moiety.
- L may contain optionally substituted alkyl group, aryl group, alkenyl group, alkynyl group, alkoxy group, ether group, ester group, amine group, amide group, imide group,
- L is an alkyl group, specifically, butyl group, when m is 1.
- Exemplary compounds include:
- compositions comprising a complex comprising a metal, nitrilotriacetic acid (NT A) and PEG (poly(ethylene glycol)) or analogue thereof.
- Suitable metals include Ca 2+ , Co 2+ , Cu 2+ , Ni 2+ , and Zn 2+ .
- the metal is Ni 2+ .
- the composition further comprises a peptide, polypeptide, or protein comprising a poly-histidine tag (His-tag).
- the Ni-NTA-PEG complex is non-covalently (i.e., reversibly) bound to the peptide, polypeptide or protein.
- Suitable peptides, polypeptides, or proteins useful in the methods described herein include any peptides, polypeptides, or proteins with a poly-histidine tag and could be readily identified by the skilled artisan.
- suitable proteins can be classified based on their molecular mechanism of activity, e.g., (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin.
- Other exemplary proteins include engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics.
- proteins useful in the invention include therapeutic proteins such as antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics.
- Suitable therapeutic proteins include proteins or peptides derived from any form of an antibody, peptide hormones, peptide ligands, signaling molecules (e.g., cytokines and chemokines), receptors, transport proteins, structural (fibrous) proteins (e.g., collagen and keratin), enzymes (e.g., digestive enzymes, blood clotting enzymes, glycolytic enzymes, helicase enzymes, aromatase enzymes, kinases, reductases, lyases, hydrolases, isomerases, ligases, transferases, antioxidants or any protein known or believed to exert a therapeutically beneficial effect.
- signaling molecules e.g., cytokines and chemokines
- receptors transport proteins
- structural (fibrous) proteins e.g., collagen and keratin
- structural (fibrous) proteins e.g., collagen and keratin
- enzymes e.g., digestive enzymes, blood clotting enzymes, glycolytic
- proteins include enzymes and regulatory proteins (e.g., collagenase), targeted proteins (e.g., antibodies or immunoadhesins), protein vaccines (e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA), and protein diagnostics (e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative).
- enzymes and regulatory proteins e.g., collagenase
- targeted proteins e.g., antibodies or immunoadhesins
- protein vaccines e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA
- protein diagnostics e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative).
- proteins useful in the invention include, but are not limited to, apoptotic proteins, e.g., tumor necrosis factor related apoptosis inducing ligand (TRAIL), receptors, e.g., estrogen receptor 1 (ESR-1), enzymes, e.g., isocitrate dehydrogenase 1 (IDH-1), interferons, e.g., interferon alpha 2 (IFNA-2), interleukins, e.g., interleukin 6 (IL-6), chemokine (C-C motif) ligands, e.g., CCL3, B-cell CLL/lymphoma 9 (BCL-9), quinone reductase 1 (QR-1), cyclooxygenase 1 (COX-1), BCL-2-like protein 4 (BCL2), caspase-1, beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), kelch-like ECH-associated protein 1 (KEAP1)
- the protein therapeutic is selected from the group consisting of etanercept (Enbrel ® , a TNF blocker), erythropoietin, darbepoetin alfa (Aranesp ® , an EPO analog), filgrastim (Neupogen ® or recombinant methionyl human granulocyte colony- stimulating factor (r-metHuG-CSF)) and pegfilgrastim (Neulasta ® , a PEGylated filgrastim).
- Embodiments of the protein therapeutic also include therapeutic antibodies such as Humira (adalimumab), Synagis (palivizumab),146B7-CHO (anti-IL15 antibody, see U.S. P.N.
- vectibix panitumumab
- Rituxan rituximab
- zevalin ibritumomab tiuxetan
- anti-CD80 monoclonal antibody mAb
- mAb galiximab
- anti-CD23 mAb lumiliximab
- M200 volociximab
- anti-Cripto mAb anti-BR3 mAb
- anti-IGFIR mAb Tysabri (natalizumab), Daclizumab
- humanized anti-CD20 mAb ocrelizumab
- soluble BAFF antagonist BR3-Fc
- anti-CD40L mAb anti-TWEAK mAb
- anti-IL5 Receptor mAb anti-ganglioside GM2 mAb
- anti-FGF8 mAb anti- VEGFR/Flt- 1 mAb
- anti-ganglioside GD2 mAb Actilyse
- Idiopathic Pulmonary Fibrosis Phase I Fibrogen LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-I), IMC-3G3 (anti-PDGFRa), MDX-1401 (anti- CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti- CCR2 chemokine receptor mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)
- Additional exemplary proteins for use in the methods described herein include the gene products of any of the following: ephrin Al, ephrin A2, ephrin A3, ephrin A4, ephrin A7, ephrin A8, angiopoietin 2, Tiel, TEK, beta5 integrin, beta3 integrin, alpha v intergrin, CASM (cancer-associated SM-like oncogene), c-myb, c-myc, ephrin-Al, ephrin-A3, ephrin- A5, ephrin-Bl, telomerase reverse transcriptase, K-ras, mdr-1, UPAR (urokinase-type plasminogen activator receptor), Bak, Bax alpha, Bax beta, Bax delta, Bax epsolin, bcl-w, HIF-alpha, ID1, ID
- the composition does not comprise a stabilizing agent, e.g., Tween 20, concentrated glycerol, or sucrose.
- a stabilizing agent e.g., Tween 20, concentrated glycerol, or sucrose.
- the Ni-NTA-PEG analogue comprises
- PEG alternatives water-soluble biocompatible polymers, either synthetic or natural
- PEG alternatives can include: polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylami.de, hyaluronic acid, chondroitin sulfate, carboxymethylcellulose,
- starch polyethylene oxide, polyvinyl alcohol, polyhydroxyalkyl
- kits comprising the compositions described herein.
- kits comprising a Ni-NTA-PEG analogue and a His-tagged protein.
- agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
- binding or “specific binding” is understood as having at least a 10 3 or more, preferably 10 4 or more, preferably 10 5 or more, preferably 10 6 or more preference for binding to a specific binding partner as compared to a non-specific binding partner (e.g., binding an antigen to a sample known to contain the cognate antibody).
- control or “reference” is meant a standard of comparison.
- "changed as compared to a control” sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator to be detected at a level that is statistically different than a sample from a normal, untreated, or control sample.
- Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art.
- An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., an antibody, a protein) or a substance produced by a reporter construct (e.g, ⁇ -galactosidase or luciferase). Depending on the method used for detection the amount and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.
- detecting As used herein, “detecting”, “detection” and the like are understood that an assay performed for identification of a specific analyte in a sample, e.g., an antigen in a sample or the level of an antigen in a sample.
- the amount of analyte or activity detected in the sample can be none or below the level of detection of the assay or method.
- an effective amount is meant an amount of a compound, alone or in a combination, required to ameliorate the symptoms of a disease relative to an untreated patient.
- the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
- polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
- a "nucleic acid encoding a polypeptide” is understood as any possible nucleic acid that upon (transcription and) translation would result in a polypeptide of the desired sequence.
- the degeneracy of the nucleic acid code is well understood. Further, it is well known that various organisms have preferred codon usage, etc. Determination of a nucleic acid sequence to encode any polypeptide is well within the ability of those of skill in the art.
- isolated or purified when used in reference to a polypeptide means that a naturally polypeptide or protein has been removed from its normal physiological environment (e.g., protein isolated from plasma or tissue, optionally bound to another protein) or is synthesized in a non- natural environment (e.g., artificially synthesized in an in vitro translation system or using chemical synthesis).
- an "isolated” or “purified” polypeptide can be in a cell-free solution or placed in a different cellular environment (e.g., expressed in a heterologous cell type).
- isolated when used in reference to a cell means the cell is in culture (i.e., not in an animal), either cell culture or organ culture, of a primary cell or cell line.
- Cells can be isolated from a normal animal, a transgenic animal, an animal having spontaneously occurring genetic changes, and/or an animal having a genetic and/or induced disease or condition.
- An isolated virus or viral vector is a virus that is removed from the cells, typically in culture, in which the virus was produced.
- kits are understood to contain at least one non-standard laboratory reagent for use in the methods of the invention in appropriate packaging, optionally containing instructions for use.
- the kit can further include any other components required to practice the method of the invention, as dry powders, concentrated solutions, or ready to use solutions.
- the kit comprises one or more containers that contain reagents for use in the methods of the invention; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
- Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.
- Nitrilotriacetic acid or "NT A” is referred to as the
- aminopolycarboxylic acid with the formula N(CH 2 C0 2 H) 3 ). It is a colorless solid that is used as a chelating agent, which forms coordination compounds with metal ions (chelates) such as Ca 2+ , Cu 2+ , Fe 3+ and Ni 2+ .
- metal ions chelates
- PEG polyethylene glycol and is a polyether compound with applications ranging from industry to medicine.
- PEGylated or “PEGylation” refers to the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated. PEGylation is achieved by incubation of a reactive derivative of PEG with the target molecule
- PK pharmacokinetics
- the substances of interest include pharmaceutical agents, hormones, nutrients, and toxins. It attempts to discover the fate of a drug from the moment that it is administered up to the point at which it is completely eliminated from the body.
- Pharmacokinetics describes how the body affects a specific drug after administration through the mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or
- glucuronosyltransferase enzymes glucuronosyltransferase enzymes
- Pharmacokinetic properties of drugs may be affected by elements such as the site of administration and the dose of administered drug. These may affect the absorption rate. Pharmacokinetics is often studied in conjunction with pharmacodynamics, the study of a drug's pharmacological effect on the body.
- phrases "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
- the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
- Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
- materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
- glycols such as propylene glycol
- polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
- esters such as ethyl oleate and ethyl laurate
- agar buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
- Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intracardiac, intraperotineal, intrathecal, intracranial, rectal, vaginal and/or parenteral administration.
- the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
- the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.
- the terms "prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
- plurality is understood to mean more than one.
- a plurality refers to at least two, three, four, five, or more.
- polypeptide or “peptide” as used herein is understood as two or more
- a peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural or non-natural amino acids joined by peptide bonds.
- Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acids sequences (e.g., fragments of naturally occurring proteins or synthetic polypeptide fragments).
- the peptide further includes one or more modifications such as modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
- polypeptides may be modified by either natural process, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
- Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation
- reduce or “increase” is meant to alter negatively or positively, respectively, by at least 5%.
- An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
- sample refers to a biological material that is isolated from its environment (e.g., blood or tissue from an animal, cells, or conditioned media from tissue culture) and is suspected of containing, or known to contain an analyte, such as a protein.
- a sample can also be a partially purified fraction of a tissue or bodily fluid.
- a reference sample can be a "normal” sample, from a donor not having the disease or condition fluid, or from a normal tissue in a subject having the disease or condition.
- a reference sample can also be from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only).
- a reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or therapeutic intervention to be tested or at the start of a prospective study.
- substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
- a reference amino acid sequence for example, any one of the amino acid sequences described herein
- nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
- such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
- Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
- BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.
- a "subject" as used herein refers to an organism.
- the organism is an animal.
- the subject is a living organism.
- the subject is a cadaver organism.
- the subject is a mammal, including, but not limited to, a human or non-human mammal.
- the subject is a domesticated mammal or a primate including a non- human primate. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep.
- a human subject may also be referred to as a patient.
- a "subject sample” can be a sample obtained from any subject, typically a blood or serum sample, however the method contemplates the use of any body fluid or tissue from a subject.
- the sample may be obtained, for example, for diagnosis of a specific individual for the presence or absence of a particular disease or condition.
- a subject "suffering from or suspected of suffering from” a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome.
- Methods for identification of subjects suffering from or suspected of suffering from conditions associated with diminished cardiac function is within the ability of those in the art.
- Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.
- susceptible to or “prone to” or “predisposed to” a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population.
- An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.
- treat refers to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
- the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
- compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
- Figures 1A- Figure IB depict a schematic of site-specific pseudo-PEGylation of His- tag fused protein.
- Figure 1 A depicts an illustration of PEGylating His-tagged protein with the reactive Ni-NTA-PEG such as compound 1 or compound 4.
- Ni-NTA-PEG selectively binds to His-tag, the stability and solubility of the protein immediately and significantly increases while maintaining its bioactivity as a covalently PEGylated protein.
- Ni-NTA-PEG improves pharmacokinetic and pharmacodynamic parameters while reclaiming the native protein's bioactivity. Therefore, Ni-NTA-PEG can be used to investigate therapeutic potential during the drug screening process.
- Figure IB depicts the chemical structures of methoxy PEG (PEG: polyethylene glycol, mw 5 kDa) and Ni-NTA-PEG analogs, Ni-monoNTA-PEG compound 1 and Ni-bisNTA-PEG compound 4.
- Figures 2A- Figure 2C depict graphs showing the effects of Ni-NTA-PEGs on the stability and bioactivity of TRAIL (Tumor necrosis factor-related apoptosis inducing ligand).
- Figure 2A is a size exclusion chromatography spectrum of TRAIL (100 ⁇ g/mL) and TRAIL associated with PEG, compound 1 and compound 4 at TRAIL: PEG molar ratio of 1:5 in 20 mM acetate buffer, pH 6.0.
- Figure 2B is a graph depicting time-dependent stability of TRAIL (400 ⁇ g/mL) and its mixtures relative to the stability at time 0 in 20 mM PBS (phosphate buffered saline), pH 7.4, at 37°C.
- Figure 2C is a graph depicting in vitro biological activity of TRAIL (10 _1 -10 4 ng/mL) and its mixtures on HCT 116 cells.
- TRAIL TRAIL
- TRAIL/PEG TRAIL with Ni-NTA-PEG analogues
- Figure 3B is a bar graph of Area Under the Curve (AUC) values from zero to infinity derived from the PK analysis. *p ⁇ 0.001 versus TRAIL alone and TRAIL/PEG, **p ⁇ 0.001 versus TRAIL/compound 1.
- Figure 3D is fluorescent microscopy images of apoptotic cell death in tumors from mice treated with TRAILs (nucleus stained with DAPI (4',6-diamidino-2-phenylindole); TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) positive apoptotic cells, red): Right: histological images of kidney stained using hematoxulin and eosin (H&E). Graphs represent + S.D.
- Figure 4 is a plot showing the binding of 1, 4 and PEG to TRAIL.
- 1 and 4 showed dissociation rate constants (k ⁇ of 3.54xl0 ⁇ 3 and 1.36xl0 ⁇ 3 s "1 , respectively, and the equilibrium dissociation constants (3 ⁇ 4) of 41.6 ⁇ and 17.7 ⁇ , respectively, with TRAIL.
- dissociation rate constants k ⁇ of 3.54xl0 ⁇ 3 and 1.36xl0 ⁇ 3 s "1 , respectively
- the equilibrium dissociation constants 3 ⁇ 4
- Figure 5A- Figure 5D are MALDI-TOF mass spectra.
- Figure 5A is a MALDI-TOF spectrum of PEG (calculated/measured m/z; 5000.00/5016.61).
- Figure 5B is a MALDI-TOF spectrum of compound 1 (53140.32/5418.69).
- Figure 5C is a MALDI-TOF spectrum of compound 4 (6398.83/6476.54).
- Figure 5D is a MALDI-TOF overlap of each spectrum from Figure 5A, Figure 5B and Figure 5C.
- Figure 6A- Figure 6C are graphs depicting cytotoxicities.
- Figure 6 A is a graph depicting cytotoxicities of TRAIL and its mixtures on normal fibroblast CCD-986sk cells.
- Figure 6B is a graph depicting cytotoxicities of PEG and Ni-NTA-PEG, 1 and 4, on CCD- 968sk ( Figure 6B) and colon cancer HCT-116 cells (Figure 6C).
- Figure 6C is a graph depicting cytotoxicities of PEG and Ni-NTA-PEG, 1 and 4 on colon cancer HCT-116 cells.
- PEGylation is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and
- PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule.
- the covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance.
- PEGylation can also provide water solubility to hydrophobic drugs and proteins.
- PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typically peptides, proteins, and antibody fragments, that can improve the safety and efficiency of many therapeutics. It produces alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity etc. These physical and chemical changes increase systemic retention of the therapeutic agent. Also, it can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.
- PEGylation the chemical attachment of poly (ethylene glycol) (PEG)
- PEG poly (ethylene glycol)
- site-specific PEGylation improves protein stability in vivo while minimizing the loss of activity associated with conventional random PEGylation (J. S. Kang, et al., Expert Opin. Emerg. Drugs 2009, 14, 363-380, and K. Knop,et al., Angew. Chem.
- a polyhistidine-tag is an amino acid motif in proteins that consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag.
- Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems. Bacterial cells are harvested and the resulting cell pellet is lysed. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound bivalent nickel or cobalt ions. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals.
- These resins are generally sepharose/agarose functionalized with a chelator, such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity.
- a chelator such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity.
- Ni-IDA iminodiacetic acid
- Ni-NTA nitrilotriacetic acid
- Co-CMA carboxylmethylaspartate
- the resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion.
- washing efficiency can be improved by the addition of
- Suitable peptides, polypeptides, or proteins useful in the methods described herein include any peptides, polypeptides, or proteins with a poly-histidine tag.
- suitable proteins can be classified based on their molecular mechanism of activity, e.g., (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin.
- Other exemplary proteins include engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics.
- proteins useful in the invention include therapeutic proteins such as antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics.
- Suitable therapeutic proteins include proteins or peptides derived from any form of an antibody, peptide hormones, peptide ligands, signaling molecules (e.g., cytokines and chemokines), receptors, transport proteins, structural (fibrous) proteins (e.g., collagen and keratin), enzymes (e.g., digestive enzymes, blood clotting enzymes, glycolytic enzymes, helicase enzymes, aromatase enzymes, kinases, reductases, lyases, hydrolases, isomerases, ligases, transferases, antioxidants or any protein known or believed to exert a therapeutically beneficial effect.
- signaling molecules e.g., cytokines and chemokines
- receptors transport proteins
- structural (fibrous) proteins e.g., collagen and keratin
- structural (fibrous) proteins e.g., collagen and keratin
- enzymes e.g., digestive enzymes, blood clotting enzymes, glycolytic
- proteins include enzymes and regulatory proteins (e.g., collagenase), targeted proteins (e.g., antibodies or immunoadhesins), protein vaccines (e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA), and protein diagnostics (e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative).
- enzymes and regulatory proteins e.g., collagenase
- targeted proteins e.g., antibodies or immunoadhesins
- protein vaccines e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA
- protein diagnostics e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative).
- proteins useful in the invention include, but are not limited to, apoptotic proteins, e.g., tumor necrosis factor related apoptosis inducing ligand (TRAIL), receptors, e.g., estrogen receptor 1 (ESR-1), enzymes, e.g., isocitrate dehydrogenase 1 (IDH-1), interferons, e.g., interferon alpha 2 (IFNA-2), interleukins, e.g., interleukin 6 (IL-6), chemokine (C-C motif) ligands, e.g., CCL3, B-cell CLL/lymphoma 9 (BCL-9), quinone reductase 1 (QR-1), cyclooxygenase 1 (COX-1), BCL-2-like protein 4 (BCL2), caspase-1, beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), kelch-like ECH-associated protein 1 (KEAP1)
- the protein therapeutic is selected from the group consisting of etanercept (Enbrel ® , a TNF blocker), erythropoietin, darbepoetin alfa (Aranesp ® , an EPO analog), filgrastim (Neupogen ® or recombinant methionyl human granulocyte colony- stimulating factor (r-metHuG-CSF)) and pegfilgrastim (Neulasta ® , a PEGylated filgrastim).
- Embodiments of the protein therapeutic also include therapeutic antibodies such as Humira (adalimumab), Synagis (palivizumab),146B7-CHO (anti-IL15 antibody, see U.S. P.N.
- vectibix panitumumab
- Rituxan rituximab
- zevalin ibritumomab tiuxetan
- anti-CD80 monoclonal antibody mAb
- mAb galiximab
- anti-CD23 mAb lumiliximab
- M200 volociximab
- anti-Cripto mAb anti-BR3 mAb
- anti-IGFIR mAb Tysabri (natalizumab), Daclizumab
- humanized anti-CD20 mAb ocrelizumab
- soluble BAFF antagonist BR3-Fc
- anti-CD40L mAb anti-TWEAK mAb
- anti-IL5 Receptor mAb anti-ganglioside GM2 mAb
- anti-FGF8 mAb anti- VEGFR/Flt- 1 mAb
- anti-ganglioside GD2 mAb Actilyse
- Additional exemplary proteins for use in the methods described herein include the gene products of any of the following: ephrin Al, ephrin A2, ephrin A3, ephrin A4, ephrin A7, ephrin A8, angiopoietin 2, Tiel, TEK, beta5 integrin, beta3 integrin, alpha v intergrin, CASM (cancer-associated SM-like oncogene), c-myb, c-myc, ephrin-Al, ephrin-A3, ephrin- A5, ephrin-Bl, telomerase reverse transcriptase, K-ras, mdr-1, UPAR (urokinase-type plasminogen activator receptor), Bak, Bax alpha, Bax beta, Bax delta, Bax epsolin, bcl-w, HIF-alpha, ID1, ID
- NTA Nitrilotriacetic acid
- Nitrilotriacetic acid is the aminopolycarboxylic acid with the formula N(CH 2 C0 2 H) 3 . It is a colorless solid that is used as a chelating agent, which forms coordination compounds with metal ions (chelates) such as Ca 2+ , Cu 2+ , and Fe + .
- NTA is a tripodal tetradentate trianionic ligand. This compound is used in complexometric titrations.
- a variant of NTA is used for protein isolation and purification in the His-tag method. The modified NTA is used to immobilize nickel to a solid support. This allows purification of proteins containing a tag consisting of six histidine residues at either terminus. Improving protein efficacy in vivo
- Described herein is a facile technique that allows for fast and simple efficacy testing of protein drugs in animal models by extending the half-life in the blood of any selected protein candidate without compromising bioactivity.
- This technique offers the benefits of site-specific PEGylation without time-consuming and costly chemical modification and purification processes, enabling high-throughput testing of protein drugs in vivo.
- the general concept used is to PEGylate proteins through a complementary interaction between an oligo- histidine tag (His-tag) and a Ni 2+ complex of nitrilotriacetic acid (NT A) (J. Crowe, H. et al., Methods Mol. Biol. 1994, 31, 371-38, and K. Terpe, Appl. Microbiol. Biotechnol.
- Biotechnol. 2004, 22, 440-444, and S. Lata, M. et al., J. Am. Chem. Soc. 2006, 128, 2365- 2372, and C. R. Goldsmith, et al., /. Am. Chem. Soc. 2006, 128, 418-419) utilize the selective and strong binding properties of His-tag to NTA.
- NTA- PEG analogues comprise a structure of the following Formula (I): Formula (I) wherein in Formula (I),
- NTA represents (nitrilotriacetic acid group
- R] is optionally substituted alkyl, optionally substituted alkoxy, or hydroxyl
- i 0 or a positive integer
- n is a positive integer
- M is a metal ion.
- i is a positive integer, preferably 5 or less, more preferably, 1-3, or particularly 1 or 2.
- M is a metal ion that can form a chelate with ligands such as any carboxyl or amine groups.
- M may be Ca 2+ , Co 2+ , Cu 2+ , Ni 2+ , Zn 2+ or Fe 2+ , more preferably, N 2+ or Co 2+ , or particularly Ni 2+ .
- ethylene oxide group has a molecular weight from about 1,000 to about 10,000 Da, particularly of about 5,000 Da.
- L may suitably conjugate with or coupled to at least one or more of NTA and amide which is further coupled to the optionally substituted ethylene oxide moiety.
- L may contain optionally substituted alkyl group, aryl group, alkenyl group, alkynyl group, alkoxy group, ether group, ester group, amine group, amide group, imide group,
- sulfonylgroup hydroxyl group, anhydride group, carbonyl group, carboxyl group, carbamide group, carbamate group, aldehyde group, amide group, maleimide group, disulfide group, alicyclic group, heteroalicyclic group, halide, amino acid (peptide), fatty acid, nucleotide, small proteins, PEG, PEG alternatives, sugar molecules, enzyme substrate, enzyme, polysaccharides, dendrimers, other nanoparticles, small molecules.
- L is an alkyl group, specifically, butyl group, when m is 1.
- Ri is an optionally substituted alkoxy, more specifically methoxy.
- suitable non-hydrogen substituents may be e.g. halo (F, CI, Br or I); cyano, nitro, hydroxy, optionally substituted Cl-20alkyl, optionally substituted Cl- 20alkoxy, such as optionally substituted alkyl (e.g.
- optionally substituted Cl-10 alkyl optionally substituted alkenyl or alkynyl preferably having 2 to about 20 carbon atoms such as such as allyl; optionally substituted ketones preferably having 1 to about 20 carbon atoms; optionally substituted alkylthio preferably having 1 to about 20 carbon atoms; optionally substituted alkylsulfinyl preferably 1 to about 20 carbon atoms; optionally substituted alkylsulfonyl preferably having 1 to about 20 carbon atoms; optionally substituted carboxy preferably have 1 to about 20 carbon atoms (which includes groups such as -COOR' where R' is H or Cl-8alkyl, including esters that are substantially non-reactive with photoacid); optionally substituted alkaryl such as optionally substituted benzyl, optionally substituted carbocyclic aryl such as optionally substituted phenyl, naphthyl, acenaphthyl, or optionally substituted heteroalicyclic or heteroaro
- NTA-PEG analogue compounds as described above and other materials used for synthesis thereof may be optionally substituted.
- a "substituted" substituent may be substituted at one or more available positions, typically 1, 2, or 3 positions by one or more suitable groups such as e.g. halogen (particularly F, CI or Br); cyano; nitro; Ci-8 alkyl; Ci_8 alkoxy; Ci_8 alkylthio; Ci_8 alkylsulfonyl; C 2 _s alkenyl; C 2 _8 alkynyl; hydroxyl; nitro; alkanoyl such as a Ci_6 alkanoyl e.g.
- PEG alternatives water-soluble biocompatible polymers, either synthetic or natural
- PEG polyethylene oxide
- PEO polyvinyl alcohol
- polyhydroxyethyl methacrylate polyacrylaniide
- hyaluronic acid chondroitin sulfate
- carboxymethylcellulose starch
- polyethylene oxide polyvinyl alcohol
- polyhydroxyalkyl (meth)acrylate polyacrylamide
- star polymers polystyrenes
- polypropylenes polymethacrylates
- polyacrylates poly ethylenes, polyethylene oxides, polysilicates
- polycarbonates polytetrafluoroethylene, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acids, poly hydroxy acids, polyesters, polycapralactone, polyhydroxybutyrate, polyphosphazenes, polyorthoesters, polyurethanes, and combinations thereof.
- Exemplary mono-NTA-PEG analogues may include
- NTA-PEG analogue compounds of the invention can be readily prepared.
- a preferred exemplary synthesis is shown in the following Scheme 1 :
- mono-NTA PEG analogues 1 may be suitably prepared.
- Scheme 1 shows the synthesis of Ni-NTA-PEG analogs, i) PEG-NHS in MeOH containing 4- methylmorpholine; ii) 2-iminothiolane hydrochloride in distilled water, NaHC03; iii) bis- maleimide amine in PBS; iv) PEG-NHS in MeOH containing 4-methylmorpholine.
- a reactive PEG group can be coupled to NTA analogs, followed by chelation with metal ion in a solution.
- exemplary mono-NTA PEG analogues can be synthesized by reacting methoxy PEG N-hydroxylsuccinimide ester (PEG-NHS) and NTA- Lys, N a ,Np-bis(carboxymethyl)-L-lysine, followed by chelation with Ni 2+ in NiCl 2 solution.
- bis-NTA PEG analogues 4 may be suitably prepared.
- Exemplary synthesis of the bis-NTA PEG analogues may include a combination of commercially available compounds including NTA-Lys, Traut's reagent (2-iminothiolane), bis-maleimide amine and PEG-NHS, without the need for harsh protection/deprotection schemes.
- a sulfhydryl group is first introduced to NTA-Lys by incubating NTA-Lys with the amine-reactive Traut's reagent; iii) thiolated NTA-Lys, NTA- Lys-SH 2, is then reacted with a bis-maleimide amine to produce bisNTA analogue 3; and iv) the thus prepared bis-NTA analogue 3 is finally conjugated with a reactive PEG group (PEG- NHS) and labeled or chelated with Ni 2+ to produce the bis-NTA PEG analogue 4.
- a methoxy PEG with a molecular weight of 5 kDa is used as a backbone.
- compositions and methods described herein allow for biologies with improved efficacy and reduced dosing profiles and toxicity compared to protein drugs. This current platform contributes to the development of biologies by reducing the cost of drug screening and streamlining evaluation in animal models.
- Compound 1 was synthesized by reacting NTA-Lys, N a ,N a -Bis(carboxymethyl)-L- lysine hydrate, (15.7 mg, 0.06 mmol) (Sigma-Aldrich, St. Louis, MO, USA) with methoxy PEG N-hydroxysuccinimide ester (PEG-NHS, mw 5 kDa) (Sigma-Aldrich) (200 mg, 0.04 mmol) in MeOH (4 mL) containing 60 ⁇ L ⁇ of 4-methylmorpholine for 6 h at room temperature. The product was precipitated by adding diethyl ether (40 mL) and vacuum dried to yield 180 mg (85%), followed by incubation in 2M NiCl 2 solution to result in 1.
- NTA-Lys 500 mg, 1.9 mmol was dissolved in distilled water (5 mL), together with NaHC0 3 (500 mg, 6 mmol) and 2-Iminothiolane hydrochloride (500 mg, 3.6 mmol) (Pierce Biotechnilogy, Inc., Rockfod, IL, USA). After heating for 15 h at 72°C, the solution was cooled, acidified to pH 3 with AcOH (0.5 mL), and concentrated under reduced pressure. The crude product was crystallized in absolute EtOH as a light beige hydroscopic solid, which was filtered, washed with absolute EtOH and dried under vacuum to give 488 mg (64 ) of NTA-Lys-SH 2.
- 6xHis-ILZ-hTRAIL 6xHis-ILZ-hTRAIL (114-281) was transfected in Escherichia coli (E. coli) using a pET23dw-His-ILZ-hTRAIL expression vector (S. Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729). Briefly, after amplification in E. coli, TRAIL expression was induced using a isopropyl-L-thio-B-D-galactopyranoside (1 mM, 7 h at 27°C).
- TRAIL was purified by Ni-affinity chromatography following stepwise washing with 50 and 100 mM imidazole buffer and eluted by 500 mM imidazole buffer. TRAIL was purified and obtained by size exclusion chromatography.
- TRAIL Interaction of TRAIL and Ni-NTA-PEGs was monitored by SEC.
- TRAIL/Ni-NTA-PEGs To prepare TRAIL/Ni-NTA-PEGs, purified TRAIL dissolved in 20 mM acetate buffer (100 mM NaCl, pH 6.0) was mixed with the same volume of acetate buffer containing 5 excess molar ratio of 1 or 4 and incubated for 1 h at room temperature. TRAIL/PEG was prepared.
- TRAIL protein (0.1 mg/mL) was immobilized on 0.05 M N-hydroxy-succinimide (NHS)- and 0.2 M N-ethyl-N' (dimethylaminopropyl) carbodiimide (EDC)-activated CM 5 chip (GE Healthcare) at a flow rate of 10 ⁇ 7 ⁇ for 7 minutes.
- a BIAcore 1000 (GE Healthcare) was used for SPR experiments at 27°C.
- PEG alone, monoNTA or bisNTA (0.2 ⁇ ) were injected onto the TRAIL immobilized surface at a flow rate of 5 ⁇ / ⁇ for 10 minutes.
- Chip surface was regenerated by injecting 200 mM EDTA at a flow rate of 10 ⁇ / ⁇ for 2 minutes followed by 5 mM NaOH. Experiments were done in degassed phosphate buffered saline buffer (pH 7.4) with 0.005% Tween 20 (BioRad).
- TRAIL formulas were determined by performing MTT assays after incubation of TRAIL, TRAIL/PEG and TRAIL/1 and 4 in both human HCT116 colon tumor cells (American Type Culture Collection, Manassas, VA, USA) and CCD-986sk normal skin fibroblasts (Korean Cell Line Bank, Seoul, Korea). Both cell lines were maintained in DMEM supplemented with 10% fetal bovine serum containing 1%
- TRAIL formulas were investigated in rats following intravenous administration.
- Male Sprague-Dawley rats (average body weight, 200 g) were cannulated in the jugular vein a day before the experiments. Animals were randomly divided to four groups and each group was administered 100 ⁇ g kg "1 (protein base) of TRAIL formulas.
- Blood samples were collected from the cannulated jugular vein at different time points (2, 5, 10, 30, 45 min and 1, 1.5, 2, 4, 6, and 12 h) after intravenous injection, and plasma were obtained by centrifugation and stored at -70°C until required for the assay.
- Active TRAIL concentrations in rat plasma were measured using commercial TRAIL ELISA kits (BioSource International, Inc.). Pharmacokinetic parameters were calculated from plasma concentration profiles by using non-compartment model analysis.
- Tumor cell apoptosis in vivo was investigated after TRAIL treatment in HCT116 tumor-bearing mice.
- tumor tissues were recovered from euthanized animals.
- Formalin-fixed, paraffin-embedded tissue blocks were sectioned (5 ⁇ ) and apoptotic cell death in tumor tissues was visualized by performing TdT-mediated dUTP nick end labeling (TUNEL) assays using a commercial kit (In situ Cell Death
- kidney tissues were harvested at the end of the experiment, fixed with 10% formalin and blocked by paraffin. Cross-sliced sections of the kidneys were stained with hematoxylin and eosin (H&E) and examined under a light microscopy.
- H&E hematoxylin and eosin
- TRAIL/1 and TRAIL/4 exhibited increasing hydrodynamic radii due to the interaction of TRAIL with the NTA appendage possessing high affinity towards H 6 .
- TRAIL/4 showed a similar SEC profile compared to that of covalently bound N-terminal PEGylated TRAIL- PEG 5 K without free TRAIL at a feed molar ratio of TRAIL: 4 above 1:5.
- TRAIL/1 failed to reach complete complexation at any ratio.
- TRAIL/Ni-NTA-PEGs After validation of TRAIL/Ni-NTA-PEGs in vitro, the PK of TRAIL/1, TRAIL/4 and TRAIL-PEG 5K , TRAIL covalently conjugated with the same molecular weight PEG, in rats after intravenous (IV) injection was analyzed. Total active TRAIL plasma levels were measured by enzyme-linked immunosorbent assay (ELISA). All PK parameters are summarized in Table 1 and illustrated in Figure 3A and Figure 6.
- TRAIL has a short half-life of 5-10 minutes in rat, mainly through rapid renal clearance (S. K. Kelley, et al., /. Pharmacol. Exp. Ther. 2001, 299, 31- 38).
- intravenously injected TRAIL and TRAIL/PEG were rapidly eliminated from rats within one hour ( Figure 3A).
- TRAIL/4 showed a prolonged elimination half-life and maintained activity up to 6 hours post-injection.
- TGI tumor growth inhibition
- TdT-mediated dUTP nick end labelling (TUNEL) assays Figure 3D.
- TRAIL/4 treated tissues demonstrated increased tumor cell apoptosis compared to those of native TRAIL and the other formulas.
- TGI TdT-mediated dUTP nick end labelling
- any injected dose of native TRAIL 50-1000 ⁇ g per mouse
- TGI 50% was not achieved under the experimental conditions.
- the major human adverse event related to high PEG exposure is renal toxicity (C. Krejsa, et al., Nat. Rev. Drug Discov 2006, 5, 507-521)
- acute renal toxicity of 4 was examined by histological investigation of renal tissues after PD studies.
- TRAIL/4 demonstrated similar antitumor efficacy to TRAILPEG5K in the same tumor model. This likely due to the different in vivo bioactivities between TRAIL-PEG5K and TRAIL/4; TRAILPEG5K has reduced activity (50% versus TRAIL) for all time points, whereas TRAIL/4 can fully recover its bioactivity once the Ni-NTAPEG is released from it in the blood. Because the administration route (IP versus IV) and dosing profiles are different, the results cannot be directly compared. Note that the technique offers all of the benefits of site-specific
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Abstract
The present invention provides compositions and methods for improving half- life of therapeutic proteins.
Description
REVERSIBLE PEGYLATION FOR SCREENING OF THERAPEUTIC
PROTEINS IN VIVO
RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.
Provisional Application No: 62,008,256, filed June 5, 2014, which is incorporated herein by reference in its entirety.
GOVERNMENT SUPPORT
This work was supported by grant number R00EB013450 awarded by the National Institute of Biomedical Imaging and Bioengineering, and by grant numbers U54CA151838 and T32EB013450 awarded by the National Cancer Institute. The government has certain rights in the invention.
BACKROUND OF THE INVENTION
A number of proteins with high therapeutic potential are identified every year. Once a potent protein is verified at the molecular and cellular levels, the next step towards clinical trials includes validation in an appropriate animal model. In the field of oncology, for example, the antitumor activity of a protein identified in vitro must be validated in tumor- bearing animal models in order to attract clinical interest. Unfortunately, most candidate protein drugs are inadequate for direct testing in a high-throughput fashion in vivo because of inherently short biological half-lives due to, e.g., non-specific proteolysis and renal clearance. Thus, many proteins with short half-lives do not exhibit similar potency in vivo as they do in vitro. As such, prior to the invention described herein, there was a pressing need in the art to identify new methods to improve protein stability in vivo.
SUMMARY OF THE INVENTION
This invention is based, in part, on the use of the complementary interaction of a His- tag and a Ni2+ complex of nitrilotriacetic acid (NT A) for quick therapeutic protein screening in vivo. Particularly, this specific and strong interactive pair is used to improve potency of therapeutic proteins in vivo after administration, e.g., systemic administration.
Described herein is the PEGylation of proteins through complementary interactions between an oligohistidine tag (His-tag) and a Ni2+ complex of nitrilotriacetic acid (NT A) to improve the half-life of therapeutic proteins in the blood following administration, e.g.,
systemic administration, in vivo. Described herein are animal models that show that this site- specific modification improves the efficacy of a modified model protein. As described in detail below, the fast modification is used to screen therapeutic proteins before applying time- consuming, expensive half-life extension technologies, e.g., site-specific PEGylation.
Provided herein are methods of improving in vitro and in vivo pharmacokinetic parameters of a peptide, a polypeptide, or a protein are carried out by coupling (i.e., fusing or conjugating) a poly-histidine tag (His-tag) to the peptide, mixing the His-tagged polypeptide with a complex comprising a metal, nitrilotriacetic acid (NT A) and PEG (poly (ethylene glycol)) or analogue thereof, thereby producing a His-tagged peptide/ Ni-NTA-PEG composition and improving in vitro and in vivo pharmacokinetic parameters.
Optionally, the method further comprises administering the composition to a subject, e.g., a human subject. In some cases, the His-tag comprises six or eight histidine amino acids. For example, the His-tag is located at the N terminus. Alternatively, the His-tag is located at the C terminus.
In some cases, a composition of the invention is administered intravenously or systemically. Other modes of administration include oral, rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Compositions comprising a composition of the invention can be added to a physiological fluid, such as blood. Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule. Parenteral modalities (subcutaneous or intravenous) may be preferable for more acute illness, or for therapy in patients that are unable to tolerate enteral administration due to gastrointestinal intolerance, ileus, or other concomitants of critical illness. Inhaled therapy may be most appropriate for pulmonary vascular diseases (e.g., pulmonary hypertension).
The compositions and methods described herein improve in vitro and in vivo pharmacokinetic and pharmacodynamics properties. For example, the Ni-NTA-PEG analogue/His -tagged protein results in a prolonged half-life, increased bioavailability, decreased clearance, and reduced aggregation of the His-tagged protein. In some cases, the improvement in efficacy comprises an increase in stability of the peptide, polypeptide, or protein in solution by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100%. The compositions and methods described herein increase the half-life of the peptide, polypeptide, or protein by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The compositions and methods described herein increase the bioavailability of the peptide, polypeptide, or protein by at least 1.5 fold, e.g., 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold. The compositions and methods described herein increase the aqueous solubility of the peptide, polypeptide, or protein by at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Preferably, the compositions and methods described herein are utilized to retain at least 1%, e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the bioactivity of the peptide, polypeptide, or protein.
Preferably, the His-tagged peptide/ Ni-NTA-PEG compositions are produced within about 30 minutes, e.g., within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, or within about 12 hours.
In one aspect, NTA-PEG analogues are provided and comprise a structure of the following Formula (I):
-ooc
,N . -coo-
NTA represents COO" (nitrilotriacetic acid group);
Ri is optionally substituted alkyl, optionally substituted alkoxy, or hydroxyl;
i is 0 or a positive integer;
Lis a linker group;
m is a positive integer; and
M is a metal ion.
Preferably, i is 1 or 2.
Preferably, M
Da to about 100 kDa, and n may have a linear or branched shape as being bound to at least one or more of NTA.
Preferably, m is 1 or 2.
In one aspect, wherein M is Ca2+, Co2+, Cu2+, Ni2+, or Zn2+.
Preferably, L may suitably conjugate with or coupled to at least one or more of NTA and amide which is further coupled to the optionally substituted ethylene oxide moiety. L may contain optionally substituted alkyl group, aryl group, alkenyl group, alkynyl group, alkoxy group, ether group, ester group, amine group, amide group, imide group,
sulfonylgroup, hydroxyl group, anhydride group, carbonyl group, carboxyl group, carbamide group, carbamate group, aldehyde group, amide group, maleimide group, disulfide group, alicyclic group, heteroalicyclic group, halide, amino acid (peptide), fatty acid, nucleotide, small proteins, PEG, PEG alternatives, sugar molecules, enzyme substrate, enzyme, polysaccharides, dendrimers, other nanoparticles, small molecules. For example, L is an alkyl group, specifically, butyl group, when m is 1.
Exemplary compounds include:
Also provided are compositions comprising a complex comprising a metal, nitrilotriacetic acid (NT A) and PEG (poly(ethylene glycol)) or analogue thereof. Suitable metals include Ca2+, Co2+, Cu2+, Ni2+, and Zn2+. Preferably, the metal is Ni2+. Optionally, the composition further comprises a peptide, polypeptide, or protein comprising a poly-histidine tag (His-tag). For example, the Ni-NTA-PEG complex is non-covalently (i.e., reversibly) bound to the peptide, polypeptide or protein.
Suitable peptides, polypeptides, or proteins useful in the methods described herein include any peptides, polypeptides, or proteins with a poly-histidine tag and could be readily identified by the skilled artisan. In some cases, suitable proteins can be classified based on their molecular mechanism of activity, e.g., (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Other exemplary proteins include engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics. For example, proteins useful in the invention include therapeutic proteins such as antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Other suitable therapeutic proteins include proteins or peptides derived from any form of an antibody, peptide hormones, peptide ligands, signaling molecules (e.g., cytokines and chemokines), receptors, transport proteins, structural (fibrous) proteins (e.g., collagen and keratin), enzymes (e.g., digestive enzymes, blood clotting enzymes, glycolytic enzymes, helicase enzymes, aromatase enzymes, kinases, reductases, lyases, hydrolases, isomerases, ligases, transferases, antioxidants or any protein known or believed to exert a therapeutically beneficial effect.
Other groups of proteins include enzymes and regulatory proteins (e.g., collagenase), targeted proteins (e.g., antibodies or immunoadhesins), protein vaccines (e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA), and protein diagnostics (e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative).
For example, proteins useful in the invention include, but are not limited to, apoptotic proteins, e.g., tumor necrosis factor related apoptosis inducing ligand (TRAIL), receptors, e.g., estrogen receptor 1 (ESR-1), enzymes, e.g., isocitrate dehydrogenase 1 (IDH-1), interferons, e.g., interferon alpha 2 (IFNA-2), interleukins, e.g., interleukin 6 (IL-6), chemokine (C-C motif) ligands, e.g., CCL3, B-cell CLL/lymphoma 9 (BCL-9), quinone
reductase 1 (QR-1), cyclooxygenase 1 (COX-1), BCL-2-like protein 4 (BCL2), caspase-1, beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), kelch-like ECH-associated protein 1 (KEAP1), and papain-like protease 2 (PLP-2).
In some embodiments, the protein therapeutic is selected from the group consisting of etanercept (Enbrel®, a TNF blocker), erythropoietin, darbepoetin alfa (Aranesp®, an EPO analog), filgrastim (Neupogen® or recombinant methionyl human granulocyte colony- stimulating factor (r-metHuG-CSF)) and pegfilgrastim (Neulasta®, a PEGylated filgrastim). Embodiments of the protein therapeutic also include therapeutic antibodies such as Humira (adalimumab), Synagis (palivizumab),146B7-CHO (anti-IL15 antibody, see U.S. P.N.
7,153,507), vectibix (panitumumab), Rituxan (rituximab), zevalin (ibritumomab tiuxetan), anti-CD80 monoclonal antibody (mAb) (galiximab), anti-CD23 mAb (lumiliximab), M200 (volociximab), anti-Cripto mAb, anti-BR3 mAb, anti-IGFIR mAb, Tysabri (natalizumab), Daclizumab, humanized anti-CD20 mAb (ocrelizumab), soluble BAFF antagonist (BR3-Fc), anti-CD40L mAb, anti-TWEAK mAb, anti-IL5 Receptor mAb, anti-ganglioside GM2 mAb, anti-FGF8 mAb, anti- VEGFR/Flt- 1 mAb, anti-ganglioside GD2 mAb, Actilyse® (alteplase), Metalyse® (tenecteplase), CAT-3888 and CAT-8015 (anti-CD22 dsFv-PE38 conjugates), CAT-354 (anti-IL13 mAb), CAT-5001 (anti-mesothelin dsFv-PE38 conjugate), GC-1008 (anti-TGF-β mAb), CAM-3001 (anti-GM-CSF Receptor mAb), ABT-874 (anti-IL12 mAb), Lymphostat B (Belimumab; anti-BlyS mAb), HGS-ETR1 (mapatumumab; human anti- TRAIL Receptor- 1 mAb), HGS-ETR2 (human anti-TRAIL Receptor-2 mAb), ABthrax™ (human, anti-protective antigen (from B. anthracis) mAb), MYO-029 (human anti-GDF-8 mAb), CAT-213 (anti-eotaxinl mAb), Erbitux, Epratuzumab, Remicade (infliximab; anti- TNF mAb), Herceptin® (traztusumab), Mylotarg (gemtuzumab ozogamicin), VECTIBLIX (panatumamab), ReoPro (abciximab), Actemra (anti-IL6 Receptor mAb), Avastin, HuMax- CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFr (zalutumumab), HuMax- Inflam, R1507 (anti-IGF-lR mAb), HuMax HepC, HuMax CD38, HuMax-TAC (anti-IL2Ra or anti-CD25 mAb), HuMax-ZP3 (anti-ZP3 mAb), Bexxar (tositumomab), Orthoclone OKT3 (muromonab-CD3), MDX-010 (ipilimumab), anti-CTLA4, CNTO 148 (golimumab; anti- TNFa Inflammation mAb), CNTO 1275 (anti-IL12/IL23 mAb), HuMax-CD4
(zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFR (zalutumumab), MDX-066 (CDA-I) and MDX-1388 (anti-C. difficile Toxin A and Toxin B C mAbs), MDX-060 (anti- CD30 mAb), MDX-018, CNTO 95 (anti-integrin receptors mAb), MDX-1307 (anti-Mannose Receptor/hCG mAb), MDX-1100 (anti-IPIO Ulcerative Colitis mAb), MDX-1303
(Valortim™), anti-B. anthracis Anthrax, MEDI-545 (MDX-1103, anti-IFNa), MDX-1106
(ONO-4538; anti-PDl), NVS Antibody #1, NVS Antibody #2, FG-3019 (anti-CTGF
Idiopathic Pulmonary Fibrosis Phase I Fibrogen), LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-I), IMC-3G3 (anti-PDGFRa), MDX-1401 (anti- CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti- CCR2 chemokine receptor mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRl fused to IgGl Fc), Zenapax (Daclizumab), Avastin (Bevacizumab), MabThera (Rituximab), MabTheraRA (Rituximab), Tarceva (Erlotinib), Zevalin (ibritumomab tiuxetan), Zetia (ezetimibe), Zyttorin (ezetimibe and simvastatin), Atacicept (TACI-Ig), NI-0401 (anti-CD3 in Crohn's disease),
Adecatumumab, Golimumab (anti-TNFa mAb), Epratuzumab, Gemtuzumab, Raptiva (efalizumab), Cimzia (certolizumab pegol, CDP 870), (Soliris) Eculizumab, Pexelizumab (Anti-C5 Complement), MED 1-524 (Numax), Lucentis (Ranibizumab), 17- IA (Panorex), Trabio (lerdelimumab), TheraCim hR3 (Nimotuzumab), Omnitarg (Pertuzumab), Osidem (IDM-I), OvaRex (B43.13), Nuvion (visilizumab), and Cantuzamab.
Additional exemplary proteins for use in the methods described herein include the gene products of any of the following: ephrin Al, ephrin A2, ephrin A3, ephrin A4, ephrin A7, ephrin A8, angiopoietin 2, Tiel, TEK, beta5 integrin, beta3 integrin, alpha v intergrin, CASM (cancer-associated SM-like oncogene), c-myb, c-myc, ephrin-Al, ephrin-A3, ephrin- A5, ephrin-Bl, telomerase reverse transcriptase, K-ras, mdr-1, UPAR (urokinase-type plasminogen activator receptor), Bak, Bax alpha, Bax beta, Bax delta, Bax epsolin, bcl-w, HIF-alpha, ID1, ID2B, ID4, IGF1, PDGFA, PDGFRA, PDGFRB, TGFbetal, TGFbeta2, TGFbeta3, TGFbetaR2, TCFbeta3, EGFR, ERBB2, ERBB3, ERBB4, FGF10, FGF11, FGF12, FGF13, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF4, FGF6, FGF7, FGF9, FGFRl, FGFR4, MT2MMP, MT3MMP, MT4MMP, MT5MMP, MT6MMP, MTMMP, FGF1, FGF2, FGF23, FGF3, FGF5, FGF8, FGFR2, FGFR3, HGF, FAQ, ID3, IGF2, IGF2R, IGF1R, PDGFB, TGFbetaRl, Flkl, Fltl, Flt4, KDR, MMP1, MMP10, MMP11, MMP12, MMP14, MMP15, MMP16, MMP17, MMP2, MMP3, MMP8, MMP9, PKC alpha, PKC beta, PKC delta, PKC eta, PKC epsilon, PKC iota, PKC mu, PKC nu, PKC tau, PKC zeta, VEGF, VEGF B, B VEGF, VEGP C, VEGF D, E2F, EBER-1, EBER-2, NS2, NS4A, NS4B, NS5A, NS5B, NS3, stmn cell factor, TGFalpha, GD3 synthase, FGF14, gag (HIV), TARBP2 and TAT (HIV).
In addition to NTA, other preferable Ni chelators for His-tag binding include iminodiacetic acid (IDA), tris(carboxymethyl)ethylene diamine (TED), and
carboxymethylated aspartate chelated with Co2+.
Preferably, the composition does not comprise a stabilizing agent, e.g., Tween 20, concentrated glycerol, or sucrose.
For example, the Ni-NTA-PEG analogue comprises
PEG alternatives (water-soluble biocompatible polymers, either synthetic or natural) can include: polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylami.de, hyaluronic acid, chondroitin sulfate, carboxymethylcellulose,
starch, polyethylene oxide, polyvinyl alcohol, polyhydroxyalkyl
(meth)acrylate,polyacrylamide, star polymers, polystyrenes, polyethylene vinyl acetates, polypropylenes, polymethacrylates, polyacrylates, polyethylenes, polyethylene oxides, polysilicates, polycarbonates, polytetrafluoroethylene, fluorocarbons, nylon, silicon rubber, poly anhydrides, polyglycolic acids, polyhydroxyacids, polyesters, polycapralactone, polyhydroxybutyrate, polyphosphazenes, polyorthoesters, polyurethanes, and combinations thereof.
The combinatorial effects of PEG molecular weights (1-100 kDa) and shapes (linear, branched) bound to NTA (1 or more) are optimized for protein pharmacological behaviors.
Also provided are kits comprising the compositions described herein. For example, provided are kits comprising a Ni-NTA-PEG analogue and a His-tagged protein.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
As used herein, "binding" or "specific binding" is understood as having at least a 103 or more, preferably 104 or more, preferably 105 or more, preferably 106 or more preference for binding to a specific binding partner as compared to a non-specific binding partner (e.g., binding an antigen to a sample known to contain the cognate antibody).
By "control" or "reference" is meant a standard of comparison. As used herein, "changed as compared to a control" sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator to be detected at a level that is statistically different than a sample from a normal, untreated, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art. An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., an antibody, a protein) or a substance produced by a reporter construct (e.g, β-galactosidase or luciferase). Depending on the method used for detection the amount and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.
As used herein, "detecting", "detection" and the like are understood that an assay performed for identification of a specific analyte in a sample, e.g., an antigen in a sample or the level of an antigen in a sample. The amount of analyte or activity detected in the sample can be none or below the level of detection of the assay or method.
By the terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by "an
effective amount" is meant an amount of a compound, alone or in a combination, required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. As used herein, a "nucleic acid encoding a polypeptide" is understood as any possible nucleic acid that upon (transcription and) translation would result in a polypeptide of the desired sequence. The degeneracy of the nucleic acid code is well understood. Further, it is well known that various organisms have preferred codon usage, etc. Determination of a nucleic acid sequence to encode any polypeptide is well within the ability of those of skill in the art.
As used herein, "isolated" or "purified" when used in reference to a polypeptide means that a naturally polypeptide or protein has been removed from its normal physiological environment (e.g., protein isolated from plasma or tissue, optionally bound to another protein) or is synthesized in a non- natural environment (e.g., artificially synthesized in an in vitro translation system or using chemical synthesis). Thus, an "isolated" or "purified" polypeptide can be in a cell-free solution or placed in a different cellular environment (e.g., expressed in a heterologous cell type). The term "purified" does not imply that the polypeptide is the only polypeptide present, but that it is essentially free (about 90-95%, up to 99-100% pure) of cellular or organismal material naturally associated with it, and thus is distinguished from naturally occurring polypeptide. Similarly, an isolated nucleic acid is removed from its normal physiological environment. "Isolated" when used in reference to a cell means the cell is in culture (i.e., not in an animal), either cell culture or organ culture, of a primary cell or cell line. Cells can be isolated from a normal animal, a transgenic animal, an animal having spontaneously occurring genetic changes, and/or an animal having a genetic and/or induced disease or condition. An isolated virus or viral vector is a virus that is removed from the cells, typically in culture, in which the virus was produced.
As used herein, "kits" are understood to contain at least one non-standard laboratory reagent for use in the methods of the invention in appropriate packaging, optionally containing instructions for use. The kit can further include any other components required to practice the method of the invention, as dry powders, concentrated solutions, or ready to use
solutions. In some embodiments, the kit comprises one or more containers that contain reagents for use in the methods of the invention; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.
As used herein, "Nitrilotriacetic acid" or "NT A" is referred to as the
aminopolycarboxylic acid with the formula N(CH2C02H)3). It is a colorless solid that is used as a chelating agent, which forms coordination compounds with metal ions (chelates) such as Ca2+, Cu2+, Fe3+ and Ni2+.
As used herein, "obtaining" is understood herein as manufacturing, purchasing, or otherwise coming into possession of.
As used herein, "PEG" is understood as polyethylene glycol and is a polyether compound with applications ranging from industry to medicine.
As used herein, "PEGylated" or "PEGylation" refers to the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated. PEGylation is achieved by incubation of a reactive derivative of PEG with the target molecule
The term, "pharmacokinetics" or "PK" refers to the branch is a branch of
pharmacology dedicated to determining the fate of substances administered externally to a living organism. The substances of interest include pharmaceutical agents, hormones, nutrients, and toxins. It attempts to discover the fate of a drug from the moment that it is administered up to the point at which it is completely eliminated from the body.
Pharmacokinetics describes how the body affects a specific drug after administration through the mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or
glucuronosyltransferase enzymes), and the effects and routes of excretion of the metabolites of the drug. Pharmacokinetic properties of drugs may be affected by elements such as the site of administration and the dose of administered drug. These may affect the absorption rate. Pharmacokinetics is often studied in conjunction with pharmacodynamics, the study of a drug's pharmacological effect on the body.
The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intracardiac, intraperotineal, intrathecal, intracranial, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.
As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.
A "polypeptide" or "peptide" as used herein is understood as two or more
independently selected natural or non-natural amino acids joined by a covalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural or non-natural amino acids joined by peptide bonds. Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acids sequences (e.g., fragments of naturally occurring proteins or synthetic polypeptide fragments). Optionally the peptide further includes one or more modifications
such as modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural process, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton, W.H. Freeman and Company, New York (1993);
Posttranslational Covalent Modification of Proteins, B. C. Johnson, ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
The term "reduce" or "increase" is meant to alter negatively or positively, respectively, by at least 5%. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
A "sample" as used herein refers to a biological material that is isolated from its environment (e.g., blood or tissue from an animal, cells, or conditioned media from tissue culture) and is suspected of containing, or known to contain an analyte, such as a protein. A sample can also be a partially purified fraction of a tissue or bodily fluid. A reference sample can be a "normal" sample, from a donor not having the disease or condition fluid, or from a normal tissue in a subject having the disease or condition. A reference sample can also be
from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only). A reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or therapeutic intervention to be tested or at the start of a prospective study.
By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e"3 and e"100 indicating a closely related sequence.
A "subject" as used herein refers to an organism. In certain embodiments, the organism is an animal. In certain embodiments, the subject is a living organism. In certain embodiments, the subject is a cadaver organism. In certain preferred embodiments, the subject is a mammal, including, but not limited to, a human or non-human mammal. In certain embodiments, the subject is a domesticated mammal or a primate including a non- human primate. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A human subject may also be referred to as a patient.
A "subject sample" can be a sample obtained from any subject, typically a blood or serum sample, however the method contemplates the use of any body fluid or tissue from a subject. The sample may be obtained, for example, for diagnosis of a specific individual for the presence or absence of a particular disease or condition.
A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient
number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Methods for identification of subjects suffering from or suspected of suffering from conditions associated with diminished cardiac function is within the ability of those in the art. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.
As used herein, "susceptible to" or "prone to" or "predisposed to" a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.
As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.
Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-Figure IB depict a schematic of site-specific pseudo-PEGylation of His- tag fused protein. Figure 1 A depicts an illustration of PEGylating His-tagged protein with the reactive Ni-NTA-PEG such as compound 1 or compound 4. Once a Ni-NTA-PEG selectively binds to His-tag, the stability and solubility of the protein immediately and significantly increases while maintaining its bioactivity as a covalently PEGylated protein. In vivo, Ni-NTA-PEG improves pharmacokinetic and pharmacodynamic parameters while reclaiming the native protein's bioactivity. Therefore, Ni-NTA-PEG can be used to investigate therapeutic potential during the drug screening process. All these benefits are acquired by simple incubation just before systemic treatment, without any further conjugation or purification processes. Figure IB depicts the chemical structures of methoxy PEG (PEG: polyethylene glycol, mw 5 kDa) and Ni-NTA-PEG analogs, Ni-monoNTA-PEG compound 1 and Ni-bisNTA-PEG compound 4.
Figures 2A- Figure 2C depict graphs showing the effects of Ni-NTA-PEGs on the stability and bioactivity of TRAIL (Tumor necrosis factor-related apoptosis inducing ligand). Figure 2A is a size exclusion chromatography spectrum of TRAIL (100 μg/mL) and TRAIL associated with PEG, compound 1 and compound 4 at TRAIL: PEG molar ratio of 1:5 in 20 mM acetate buffer, pH 6.0. Figure 2B is a graph depicting time-dependent stability of TRAIL (400μg/mL) and its mixtures relative to the stability at time 0 in 20 mM PBS (phosphate buffered saline), pH 7.4, at 37°C. Figure 2C is a graph depicting in vitro biological activity of TRAIL (10_1-104 ng/mL) and its mixtures on HCT 116 cells.
Cytotoxicities of formulas were determined by performing MTT (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays after incubation for 24 hours. Graph represents mean + S.D. (n=4).
Figures 3A-Figure 3D depict data showing the effects of Ni-NTA-PEGs on the pharmacological efficacy of TRAIL. Figure 3A is a graph depicting the PK
(pharmacokinetic) profiles of TRAIL, TRAIL/PEG, and TRAIL with Ni-NTA-PEG analogues. Cannulated Sprague-Dawley rats were given an intravenous injection of TRAIL (10( g/kg, based on the TRAIL concentration) and plasma concentrations were monitored by ELISA (enzyme-linked immunosorbent assay) assay (n=4). Figure 3B is a bar graph of Area Under the Curve (AUC) values from zero to infinity derived from the PK analysis. *p<0.001 versus TRAIL alone and TRAIL/PEG, **p<0.001 versus TRAIL/compound 1. Figure 3C is a graph depicting antitumor activity of TRAIL formulations in HCT116 human colon cancer- bearing mice. Tumor growth suppression was monitored while treating mice with TRAIL (15( g/mouse, based on the TRAIL concentration) by IV (intravenous) injection every 2 days starting at 5 days after tumor inoculation (n=6). *p<0.001 versus control, **/?<0.001 versus TRAIL alone and TRAIL/PEG. Figure 3D is fluorescent microscopy images of apoptotic cell death in tumors from mice treated with TRAILs (nucleus stained with DAPI (4',6-diamidino-2-phenylindole); TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) positive apoptotic cells, red): Right: histological images of kidney stained using hematoxulin and eosin (H&E). Graphs represent + S.D.
Figure 4 is a plot showing the binding of 1, 4 and PEG to TRAIL. 1 and 4 showed dissociation rate constants (k< of 3.54xl0~3 and 1.36xl0~3 s"1, respectively, and the equilibrium dissociation constants (¾) of 41.6 μΜ and 17.7μΜ, respectively, with TRAIL. When an excess amount of EDTA was treated while 4 was bound to TRAIL, quantitative dissociation was observed immediately as evidenced by significantly decreased RU value. This demonstrates that 4 binds to TRAIL selectively via the attached His -tag.
Figure 5A-Figure 5D are MALDI-TOF mass spectra. Figure 5A is a MALDI-TOF spectrum of PEG (calculated/measured m/z; 5000.00/5016.61). Figure 5B is a MALDI-TOF spectrum of compound 1 (53140.32/5418.69). Figure 5C is a MALDI-TOF spectrum of compound 4 (6398.83/6476.54). Figure 5D is a MALDI-TOF overlap of each spectrum from Figure 5A, Figure 5B and Figure 5C.
Figure 6A-Figure 6C are graphs depicting cytotoxicities. Figure 6 A is a graph depicting cytotoxicities of TRAIL and its mixtures on normal fibroblast CCD-986sk cells. Figure 6B is a graph depicting cytotoxicities of PEG and Ni-NTA-PEG, 1 and 4, on CCD- 968sk (Figure 6B) and colon cancer HCT-116 cells (Figure 6C). Figure 6C is a graph depicting cytotoxicities of PEG and Ni-NTA-PEG, 1 and 4 on colon cancer HCT-116 cells.
DETAILED DESCRIPTION OF THE INVENTION
A number of proteins with high therapeutic potential are identified every year. Once a potent protein is verified at the molecular and cellular levels, the next step towards clinical studies includes validation in an appropriate animal model. In the field of oncology, for example, the antitumor activity of a protein identified in vitro must be validated in tumor- bearing animal models in order to attract clinical interest. Unfortunately, most candidate protein drugs are inadequate for direct testing in a high-throughput fashion in vivo because of inherently short biological half-lives mainly by non-specific proteolysis and renal clearance (C. Krejsa, et al., Nat. Rev. Drug Discov 2006, 5, 507-521, and S. Frokjaer, D. E. Otzen, Nat. Rev. Drug Discov. 2005, 4, 298-306). Many proteins with short half-lives do not exhibit similar potency in vivo as they do in vitro (S. Frokjaer, D. E. Otzen, Nat. Rev. Drug Discov. 2005, 4, 298-306).
PEGylation
PEGylation is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and
macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.
PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typically peptides, proteins, and antibody fragments, that can improve the safety and efficiency of many therapeutics. It produces alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity etc. These physical and chemical changes increase systemic retention of the therapeutic agent. Also, it can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.
For decades, PEGylation, the chemical attachment of poly (ethylene glycol) (PEG), has been considered the gold standard for enhancing stability, half-life, and aqueous solubility of protein drugs (J. M. Harris, R. B. Chess, Nat. Rev. Drug Discov. 2003, 2, 214- 221 and B. Obermeier B, et a., Angew. Chem. 2011, 50, 7988-7997). In particular, site- specific PEGylation improves protein stability in vivo while minimizing the loss of activity
associated with conventional random PEGylation (J. S. Kang, et al., Expert Opin. Emerg. Drugs 2009, 14, 363-380, and K. Knop,et al., Angew. Chem. 2010, 49, 6288-6308; and G. Pasut, F. M. Veronese, J. Control Release 2012, 161, 461-472). Still, prior to the invention described herein, scientists were limited in high throughput screening tools before performing costly site-specific PEGylation to evaluate the true bioactivity of their library of protein drugs in vivo.
Polvhistidine-Tag
A polyhistidine-tag is an amino acid motif in proteins that consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag.
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems. Bacterial cells are harvested and the resulting cell pellet is lysed. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound bivalent nickel or cobalt ions. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals. These resins are generally sepharose/agarose functionalized with a chelator, such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. With Ni-based methods, washing efficiency can be improved by the addition of 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole). Generally, nickel-based resins have higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by SDS-PAGE and Western blotting.
Suitable peptides, polypeptides, or proteins useful in the methods described herein include any peptides, polypeptides, or proteins with a poly-histidine tag. In some cases, suitable proteins can be classified based on their molecular mechanism of activity, e.g., (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Other exemplary proteins include engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics. For example, proteins useful in the invention include therapeutic proteins such as antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone
morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Other suitable therapeutic proteins include proteins or peptides derived from any form of an antibody, peptide hormones, peptide ligands, signaling molecules (e.g., cytokines and chemokines), receptors, transport proteins, structural (fibrous) proteins (e.g., collagen and keratin), enzymes (e.g., digestive enzymes, blood clotting enzymes, glycolytic enzymes, helicase enzymes, aromatase enzymes, kinases, reductases, lyases, hydrolases, isomerases, ligases, transferases, antioxidants or any protein known or believed to exert a therapeutically beneficial effect.
Other groups of proteins include enzymes and regulatory proteins (e.g., collagenase), targeted proteins (e.g., antibodies or immunoadhesins), protein vaccines (e.g., a vaccine against the non-infectious protein on the outer surface of Borrelia burgdorferi, OspA), and protein diagnostics (e.g., a non- infectious protein component of Mycobacterium tuberculosis, DPPD (diagnostic purified protein derivative). A non-comprehensive list of potential therapeutic proteins useful in the methods described herein is provided in Dimitrov D, 2012 Methods Mol Biol, 899: 1-26; and Leader et al., 2008 Nat Rev Drug Discov, 7(l):21-39, each of which is incorporated herein by reference.
For example, proteins useful in the invention include, but are not limited to, apoptotic proteins, e.g., tumor necrosis factor related apoptosis inducing ligand (TRAIL), receptors, e.g., estrogen receptor 1 (ESR-1), enzymes, e.g., isocitrate dehydrogenase 1 (IDH-1), interferons, e.g., interferon alpha 2 (IFNA-2), interleukins, e.g., interleukin 6 (IL-6), chemokine (C-C motif) ligands, e.g., CCL3, B-cell CLL/lymphoma 9 (BCL-9), quinone reductase 1 (QR-1), cyclooxygenase 1 (COX-1), BCL-2-like protein 4 (BCL2), caspase-1, beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), kelch-like ECH-associated protein 1 (KEAP1), and papain-like protease 2 (PLP-2).
In some embodiments, the protein therapeutic is selected from the group consisting of etanercept (Enbrel®, a TNF blocker), erythropoietin, darbepoetin alfa (Aranesp®, an EPO analog), filgrastim (Neupogen® or recombinant methionyl human granulocyte colony- stimulating factor (r-metHuG-CSF)) and pegfilgrastim (Neulasta®, a PEGylated filgrastim). Embodiments of the protein therapeutic also include therapeutic antibodies such as Humira (adalimumab), Synagis (palivizumab),146B7-CHO (anti-IL15 antibody, see U.S. P.N.
7,153,507), vectibix (panitumumab), Rituxan (rituximab), zevalin (ibritumomab tiuxetan), anti-CD80 monoclonal antibody (mAb) (galiximab), anti-CD23 mAb (lumiliximab), M200 (volociximab), anti-Cripto mAb, anti-BR3 mAb, anti-IGFIR mAb, Tysabri (natalizumab), Daclizumab, humanized anti-CD20 mAb (ocrelizumab), soluble BAFF antagonist (BR3-Fc),
anti-CD40L mAb, anti-TWEAK mAb, anti-IL5 Receptor mAb, anti-ganglioside GM2 mAb, anti-FGF8 mAb, anti- VEGFR/Flt- 1 mAb, anti-ganglioside GD2 mAb, Actilyse® (alteplase), Metalyse® (tenecteplase), CAT-3888 and CAT-8015 (anti-CD22 dsFv-PE38 conjugates), CAT-354 (anti-IL13 mAb), CAT-5001 (anti-mesothelin dsFv-PE38 conjugate), GC-1008 (anti-TGF-β mAb), CAM-3001 (anti-GM-CSF Receptor mAb), ABT-874 (anti-IL12 mAb), Lymphostat B (Belimumab; anti-BlyS mAb), HGS-ETR1 (mapatumumab; human anti- TRAIL Receptor- 1 mAb), HGS-ETR2 (human anti-TRAIL Receptor-2 mAb), ABthrax™ (human, anti-protective antigen (from B. anthracis) mAb), MYO-029 (human anti-GDF-8 mAb), CAT-213 (anti-eotaxinl mAb), Erbitux, Epratuzumab, Remicade (infliximab; anti- TNF mAb), Herceptin® (traztusumab), Mylotarg (gemtuzumab ozogamicin), VECTIBLIX (panatumamab), ReoPro (abciximab), Actemra (anti-IL6 Receptor mAb), Avastin, HuMax- CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFr (zalutumumab), HuMax- Inflam, R1507 (anti-IGF-lR mAb), HuMax HepC, HuMax CD38, HuMax-TAC (anti-IL2Ra or anti-CD25 mAb), HuMax-ZP3 (anti-ZP3 mAb), Bexxar (tositumomab), Orthoclone OKT3 (muromonab-CD3), MDX-010 (ipilimumab), anti-CTLA4, CNTO 148 (golimumab; anti- TNFa Inflammation mAb), CNTO 1275 (anti-IL12/IL23 mAb), HuMax-CD4
(zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFR (zalutumumab), MDX-066 (CDA-I) and MDX-1388 (anti-C. difficile Toxin A and Toxin B C mAbs), MDX-060 (anti- CD30 mAb), MDX-018, CNTO 95 (anti-integrin receptors mAb), MDX-1307 (anti-Mannose Receptor/hCG mAb), MDX-1100 (anti-IPIO Ulcerative Colitis mAb), MDX-1303
(Valortim™), anti-B. anthracis Anthrax, MEDI-545 (MDX-1103, anti-IFNa), MDX-1106 (ONO-4538; anti-PDl), NVS Antibody #1, NVS Antibody #2, FG-3019 (anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen), LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-I), IMC-3G3 (anti-PDGFRa), MDX-1401 (anti- CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti- CCR2 chemokine receptor mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRl fused to IgGl Fc), Zenapax (Daclizumab), Avastin (Bevacizumab), MabThera (Rituximab), MabTheraRA (Rituximab), Tarceva (Erlotinib), Zevalin (ibritumomab tiuxetan), Zetia (ezetimibe), Zyttorin (ezetimibe and simvastatin), Atacicept (TACI-Ig), NI-0401 (anti-CD3 in Crohn's disease),
Adecatumumab, Golimumab (anti-TNFa mAb), Epratuzumab, Gemtuzumab, Raptiva
(efalizumab), Cimzia (certolizumab pegol, CDP 870), (Soliris) Eculizumab, Pexelizumab (Anti-C5 Complement), MED 1-524 (Numax), Lucentis (Ranibizumab), 17- IA (Panorex), Trabio (lerdelimumab), TheraCim hR3 (Nimotuzumab), Omnitarg (Pertuzumab), Osidem (IDM-I), OvaRex (B43.13), Nuvion (visilizumab), and Cantuzamab.
Additional exemplary proteins for use in the methods described herein include the gene products of any of the following: ephrin Al, ephrin A2, ephrin A3, ephrin A4, ephrin A7, ephrin A8, angiopoietin 2, Tiel, TEK, beta5 integrin, beta3 integrin, alpha v intergrin, CASM (cancer-associated SM-like oncogene), c-myb, c-myc, ephrin-Al, ephrin-A3, ephrin- A5, ephrin-Bl, telomerase reverse transcriptase, K-ras, mdr-1, UPAR (urokinase-type plasminogen activator receptor), Bak, Bax alpha, Bax beta, Bax delta, Bax epsolin, bcl-w, HIF-alpha, ID1, ID2B, ID4, IGF1, PDGFA, PDGFRA, PDGFRB, TGFbetal, TGFbeta2, TGFbeta3, TGFbetaR2, TCFbeta3, EGFR, ERBB2, ERBB3, ERBB4, FGF10, FGF11, FGF12, FGF13, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF4, FGF6, FGF7, FGF9, FGFR1, FGFR4, MT2MMP, MT3MMP, MT4MMP, MT5MMP, MT6MMP, MTMMP, FGF1, FGF2, FGF23, FGF3, FGF5, FGF8, FGFR2, FGFR3, HGF, FAQ, ID3, IGF2, IGF2R, IGF1R, PDGFB, TGFbetaRl, Flkl, Fltl, Flt4, KDR, MMP1, MMP10, MMP11, MMP12, MMP14, MMP15, MMP16, MMP17, MMP2, MMP3, MMP8, MMP9, PKC alpha, PKC beta, PKC delta, PKC eta, PKC epsilon, PKC iota, PKC mu, PKC nu, PKC tau, PKC zeta, VEGF, VEGF B, B VEGF, VEGP C, VEGF D, E2F, EBER-1, EBER-2, NS2, NS4A, NS4B, NS5A, NS5B, NS3, stmn cell factor, TGFalpha, GD3 synthase, FGF14, gag (HIV), TARBP2 and TAT (HIV).
Nitrilotriacetic acid (NTA)
Nitrilotriacetic acid (NTA) is the aminopolycarboxylic acid with the formula N(CH2C02H)3. It is a colorless solid that is used as a chelating agent, which forms coordination compounds with metal ions (chelates) such as Ca2+, Cu2+, and Fe +.
The uses of NTA are similar to that of EDTA, both being chelating agents. In contrast to EDTA, NTA is easily biodegradable and is almost completely removed during wastewater treatment. It is used for water softening and as a replacement to sodium and potassium triphosphate in detergents, and cleansers. NTA is a tripodal tetradentate trianionic ligand. This compound is used in complexometric titrations. A variant of NTA is used for protein isolation and purification in the His-tag method. The modified NTA is used to immobilize nickel to a solid support. This allows purification of proteins containing a tag consisting of six histidine residues at either terminus.
Improving protein efficacy in vivo
Described herein is a facile technique that allows for fast and simple efficacy testing of protein drugs in animal models by extending the half-life in the blood of any selected protein candidate without compromising bioactivity. This technique offers the benefits of site-specific PEGylation without time-consuming and costly chemical modification and purification processes, enabling high-throughput testing of protein drugs in vivo. The general concept used is to PEGylate proteins through a complementary interaction between an oligo- histidine tag (His-tag) and a Ni2+ complex of nitrilotriacetic acid (NT A) (J. Crowe, H. et al., Methods Mol. Biol. 1994, 31, 371-38, and K. Terpe, Appl. Microbiol. Biotechnol. 2003, 60, 523-533). For example, protein immobilization techniques (G. Zhen, et al., Adv. Funct. Mater. 2006, 16, 243-251and M. A. Bruckman, et al., ACS Nano 2011, 5, 1606-1616, and M. J. Ludden, et al., Angew. Chem. 2007, 46, 4104-4107, and V. Roullier, et al., Nano Lett. 2009, 9, 1228-1234) and protein labeling with fluorophores (E. G. Guignet, et al., Nat.
Biotechnol. 2004, 22, 440-444, and S. Lata, M. et al., J. Am. Chem. Soc. 2006, 128, 2365- 2372, and C. R. Goldsmith, et al., /. Am. Chem. Soc. 2006, 128, 418-419) utilize the selective and strong binding properties of His-tag to NTA.
In spite of numerous applications based on the His-tag/NTA pair, including a couple for therapeutic protein research (A. Mero, Pharm. Res. 2011, 28, 2412-2421), prior to the invention described herein, no studies had successfully utilized this specific and strong interactive pair to improve the potency of therapeutic proteins in vivo after systemic administration. As described herein, PEG analogues containing an NTA moiety were selectively labelled at specific sites of His-tagged proteins by simple mixing and
demonstrating the benefits of site-specific PEGylated proteins. Then, in vivo efficacy was tested and validated of any protein candidate in a rapid and convenient fashion. Here, with a rationally designed Ni-NTA-PEG analogue and a biologically relevant His-tagged protein, a practical technique for use in vivo is exemplified, as shown by the amplified pharmacological efficacy of the native protein.
NTA-PEG Analogues
In one aspect, NTA- PEG analogues are provided that comprise a structure of the following Formula (I):
Formula (I) wherein in Formula (I),
-ooc
R] is optionally substituted alkyl, optionally substituted alkoxy, or hydroxyl;
i is 0 or a positive integer;
Lis a linker group;
m is a positive integer; and
M is a metal ion.
Preferably, i is a positive integer, preferably 5 or less, more preferably, 1-3, or particularly 1 or 2.
Preferably, M is a metal ion that can form a chelate with ligands such as any carboxyl or amine groups. In preferred embodiments, M may be Ca2+, Co2+, Cu2+, Ni2+, Zn2+ or Fe2+, more preferably, N 2+ or Co2+, or particularly Ni2+.
Preferably,
may have a molecular weight ranging from about 100 to about 1,000,000 Da, preferably ranging from about 500 to about 500,000 Da, more preferably 1,000 to about 100,000 Da. Exemplary ethylene oxide group has a molecular weight from about 1,000 to about 10,000 Da, particularly of about 5,000 Da.
Preferably, m is 5 or less, preferably 1-4, or more preferably 1 or 2. In certain embodiments, based on m, the NTA-PEG analogue may be mono-NTA-PEG analogue (m = 1), bis-NTA-PEG analogue (m = 2), tris-NTA-PEG analogue (m = 3), or tetra-NTA-PEG analogue (m = 4).
Preferably, L may suitably conjugate with or coupled to at least one or more of NTA and amide which is further coupled to the optionally substituted ethylene oxide moiety. L may contain optionally substituted alkyl group, aryl group, alkenyl group, alkynyl group, alkoxy group, ether group, ester group, amine group, amide group, imide group,
sulfonylgroup, hydroxyl group, anhydride group, carbonyl group, carboxyl group, carbamide group, carbamate group, aldehyde group, amide group, maleimide group, disulfide group,
alicyclic group, heteroalicyclic group, halide, amino acid (peptide), fatty acid, nucleotide, small proteins, PEG, PEG alternatives, sugar molecules, enzyme substrate, enzyme, polysaccharides, dendrimers, other nanoparticles, small molecules.
In particular embodiments, L is an alkyl group, specifically, butyl group, when m is 1.
In particular embodiments, Ri is an optionally substituted alkoxy, more specifically methoxy.
In the above Formula (I), suitable non-hydrogen substituents, may be e.g. halo (F, CI, Br or I); cyano, nitro, hydroxy, optionally substituted Cl-20alkyl, optionally substituted Cl- 20alkoxy, such as optionally substituted alkyl (e.g. optionally substituted Cl-10 alkyl), optionally substituted alkenyl or alkynyl preferably having 2 to about 20 carbon atoms such as such as allyl; optionally substituted ketones preferably having 1 to about 20 carbon atoms; optionally substituted alkylthio preferably having 1 to about 20 carbon atoms; optionally substituted alkylsulfinyl preferably 1 to about 20 carbon atoms; optionally substituted alkylsulfonyl preferably having 1 to about 20 carbon atoms; optionally substituted carboxy preferably have 1 to about 20 carbon atoms (which includes groups such as -COOR' where R' is H or Cl-8alkyl, including esters that are substantially non-reactive with photoacid); optionally substituted alkaryl such as optionally substituted benzyl, optionally substituted carbocyclic aryl such as optionally substituted phenyl, naphthyl, acenaphthyl, or optionally substituted heteroalicyclic or heteroaromatic group such as pyridyl, furanyl, pyrrole, thiophene, furan, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, triazole, furanzan, oxadiazole, thiadiazole, dithiazole, terazole, pyran, thiopyran, diazine, oxazine, thiazine, dioxine, dithine, and triazine and polyaromatic groups containing one or more of such moieties.
Various moieties of NTA-PEG analogue compounds as described above and other materials used for synthesis thereof may be optionally substituted. A "substituted" substituent may be substituted at one or more available positions, typically 1, 2, or 3 positions by one or more suitable groups such as e.g. halogen (particularly F, CI or Br); cyano; nitro; Ci-8 alkyl; Ci_8 alkoxy; Ci_8 alkylthio; Ci_8 alkylsulfonyl; C2_s alkenyl; C2_8 alkynyl; hydroxyl; nitro; alkanoyl such as a Ci_6 alkanoyl e.g. acyl, haloalkyl particularly Ci_8 haloalkyl such as CF3; -CONHR, -CONRR' where R and R' are optionally substituted d_8alkyl; -COOH, COC, >C=0; and the like.
Furthermore, PEG alternatives (water-soluble biocompatible polymers, either synthetic or natural) may also be substituted for PEG like: polyethylene oxide (PEO),
polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylaniide, hyaluronic acid, chondroitin sulfate, carboxymethylcellulose, starch, polyethylene oxide, polyvinyl alcohol, polyhydroxyalkyl (meth)acrylate,polyacrylamide, star polymers, polystyrenes, polyethylene vinyl acetates, polypropylenes, polymethacrylates, polyacrylates, poly ethylenes, polyethylene oxides, polysilicates, polycarbonates, polytetrafluoroethylene, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acids, poly hydroxy acids, polyesters, polycapralactone, polyhydroxybutyrate, polyphosphazenes, polyorthoesters, polyurethanes, and combinations thereof.
Exemplary mono-NTA-PEG analogues may include
In one aspect of the invention, NTA-PEG analogue compounds of the invention can be readily prepared. A preferred exemplary synthesis is shown in the following Scheme 1 :
Scheme 1
In a preferred embodiment, mono-NTA PEG analogues 1 may be suitably prepared. Scheme 1 shows the synthesis of Ni-NTA-PEG analogs, i) PEG-NHS in MeOH containing 4- methylmorpholine; ii) 2-iminothiolane hydrochloride in distilled water, NaHC03; iii) bis- maleimide amine in PBS; iv) PEG-NHS in MeOH containing 4-methylmorpholine.
Preferably, i) a reactive PEG group can be coupled to NTA analogs, followed by chelation with metal ion in a solution. Exemplary mono-NTA PEG analogues can be synthesized by reacting methoxy PEG N-hydroxylsuccinimide ester (PEG-NHS) and NTA- Lys, Na,Np-bis(carboxymethyl)-L-lysine, followed by chelation with Ni2+ in NiCl2 solution.
In a preferred embodiment, bis-NTA PEG analogues 4 may be suitably prepared. Exemplary synthesis of the bis-NTA PEG analogues may include a combination of commercially available compounds including NTA-Lys, Traut's reagent (2-iminothiolane), bis-maleimide amine and PEG-NHS, without the need for harsh protection/deprotection schemes. In particular embodiments, ii) a sulfhydryl group is first introduced to NTA-Lys by incubating NTA-Lys with the amine-reactive Traut's reagent; iii) thiolated NTA-Lys, NTA- Lys-SH 2, is then reacted with a bis-maleimide amine to produce bisNTA analogue 3; and iv) the thus prepared bis-NTA analogue 3 is finally conjugated with a reactive PEG group (PEG- NHS) and labeled or chelated with Ni2+ to produce the bis-NTA PEG analogue 4.
Preferably, in Scheme 1 , a methoxy PEG with a molecular weight of 5 kDa is used as a backbone.
This project was initiated with the aim of introducing a facile and versatile technique that increases in vivo stability of any protein while maintaining that protein's bioactivity for in vivo applications. This technique is appropriate for performance in a high-throughput manner and nontoxic for possible clinical translation. Using TRAIL as a model protein, a unique Ni-NTA-PEG analogue associated with His-tagged protein was able to provide outstanding physicochemical stability without compromising bioactivity. Moreover, the Ni- NTA-PEG analogue not only improved the half-life of the protein, but, more importantly, maximized the pharmacological efficacy of the protein drug in vivo.
The compositions and methods described herein allow for biologies with improved efficacy and reduced dosing profiles and toxicity compared to protein drugs. This current platform contributes to the development of biologies by reducing the cost of drug screening and streamlining evaluation in animal models.
EXAMPLES
Example 1 : Materials and Methods
Synthesis of NTA-PEG analogs
Exemplary syntheses are set forth are shown in the Scheme 1 above.
Synthesis of mono-Ni-NTA-PEG analog:
Compound 1 was synthesized by reacting NTA-Lys, Na,Na-Bis(carboxymethyl)-L- lysine hydrate, (15.7 mg, 0.06 mmol) (Sigma-Aldrich, St. Louis, MO, USA) with methoxy PEG N-hydroxysuccinimide ester (PEG-NHS, mw 5 kDa) (Sigma-Aldrich) (200 mg, 0.04 mmol) in MeOH (4 mL) containing 60 μL· of 4-methylmorpholine for 6 h at room temperature. The product was precipitated by adding diethyl ether (40 mL) and vacuum dried to yield 180 mg (85%), followed by incubation in 2M NiCl2 solution to result in 1.
Compounds were characterized by and MALDI-TOF mass spectrometry (Figures 5B and 5D).
Synthesis of bis-Ni-NTA-PEG analogs:
To synthesize 2, NTA-Lys (500 mg, 1.9 mmol) was dissolved in distilled water (5 mL), together with NaHC03 (500 mg, 6 mmol) and 2-Iminothiolane hydrochloride (500 mg, 3.6 mmol) (Pierce Biotechnilogy, Inc., Rockfod, IL, USA). After heating for 15 h at 72°C, the solution was cooled, acidified to pH 3 with AcOH (0.5 mL), and concentrated under reduced pressure. The crude product was crystallized in absolute EtOH as a light beige
hydroscopic solid, which was filtered, washed with absolute EtOH and dried under vacuum to give 488 mg (64 ) of NTA-Lys-SH 2. For bisMal-NTA-Lys 3, bis-maleimide amine trifluoroacetic acid (200 mg, 0.3 mmol) (Quanta Biodesign Ltd., Powell, Ohio, USA) was dissolved in phosphate buffered saline (10 mM, pH 7.5, 3 mL) and mixed with 2 (280 mg, 0.7 mmol) in the buffer. After stirring for 1 h, the solution was concentrated under reduced pressure. The crude product was crystallized in absolute EtOH as a light beige hydroscopic solid, which was filtered, washed with absolute EtOH and dried under vacuum to give 110 mg (55 ) of 3. A slight excess of 3 (80 mg, 0.06 mmol) was dissolved in methanol (2 mL) containing 4-methylmorpholine (60 μί). Then the solution was added to PEG- NHS (200 mg, 0.04 mmol) dissolved in MeOH (2 mL) and stirred for 6 h at room temperature and purified as described above to yield 164 mg (65 ), followed by incubation in 2M NiCl2 solution to result in 4. Compounds were characterized by and MALDI-TOF mass spectrometry (Figures 5C and 5D).
Purification of TRAIL
An active TRAIL including an N-terminal His-tag and trimer-forming zipper sequence, 6xHis-ILZ-hTRAIL (114-281), was transfected in Escherichia coli (E. coli) using a pET23dw-His-ILZ-hTRAIL expression vector (S. Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729). Briefly, after amplification in E. coli, TRAIL expression was induced using a isopropyl-L-thio-B-D-galactopyranoside (1 mM, 7 h at 27°C). Harvested cells were lysed and soluble TRAIL was purified by Ni-affinity chromatography following stepwise washing with 50 and 100 mM imidazole buffer and eluted by 500 mM imidazole buffer. TRAIL was purified and obtained by size exclusion chromatography.
Size exclusion chromatography (SEC)
Interaction of TRAIL and Ni-NTA-PEGs was monitored by SEC. The relative molecular size of TRAIL, TRAIL/PEG, TRAIL/1 and TRAIL/4 were determined using a Superose 12 HR 10/30 column (GE Healthcare). The column was equilibrated with 20 mM acetate buffer (100 mM NaCl, pH 6.0) and eluted at a constant flow rate of 1 mL min 1. To prepare TRAIL/Ni-NTA-PEGs, purified TRAIL dissolved in 20 mM acetate buffer (100 mM NaCl, pH 6.0) was mixed with the same volume of acetate buffer containing 5 excess molar ratio of 1 or 4 and incubated for 1 h at room temperature. TRAIL/PEG was prepared.
Real Time Binding Experiments
TRAIL protein (0.1 mg/mL) was immobilized on 0.05 M N-hydroxy-succinimide (NHS)- and 0.2 M N-ethyl-N' (dimethylaminopropyl) carbodiimide (EDC)-activated CM 5 chip (GE Healthcare) at a flow rate of 10 μΙ7ιηίη for 7 minutes. A BIAcore 1000 (GE
Healthcare) was used for SPR experiments at 27°C. PEG alone, monoNTA or bisNTA (0.2μΜ) were injected onto the TRAIL immobilized surface at a flow rate of 5 μί/ιηίη for 10 minutes. Chip surface was regenerated by injecting 200 mM EDTA at a flow rate of 10 μί/ιηίη for 2 minutes followed by 5 mM NaOH. Experiments were done in degassed phosphate buffered saline buffer (pH 7.4) with 0.005% Tween 20 (BioRad).
In vitro stability test
The physical stabilities of samples were investigated by measuring solubility changes in 20 mM phosphate buffer (100 mM NaCl, pH 7.5) (S. Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729) . Each sample solution was prepared at a fixed concentration of 400 μg mL"1 (protein base) and incubated at 37°C. At predetermined time points (0, 10, 30 min and 1, 2, 3, 6, 12, 24 h), 100 μΕ aliquots from each sample were centrifuged at 10,000 rpm for 20 min to remove denatured protein aggregates. Protein concentration in supernatants was determined using bicinchoninic acid protein assays using bovine serum albumin (BSA) as a standard molecule.
In vitro biological activity
The in vitro cytotoxicity of TRAIL formulas were determined by performing MTT assays after incubation of TRAIL, TRAIL/PEG and TRAIL/1 and 4 in both human HCT116 colon tumor cells (American Type Culture Collection, Manassas, VA, USA) and CCD-986sk normal skin fibroblasts (Korean Cell Line Bank, Seoul, Korea). Both cell lines were maintained in DMEM supplemented with 10% fetal bovine serum containing 1%
penicillin/streptomycin in a 37°C/5% C02 incubator. For dose-dependent cytotoxicity assays, cells were seeded in 96-well plates at lxlO4 cells/well and pre-incubated for 24 h. Media were then replaced with fresh serum-free DMEM, and predetermined amounts of samples were added to the final concentrations of 0-104 ng mL"1. Cytotoxicity was determined by performing MTT assays after incubation for 24 hours.
In vivo pharmacokinetics
The pharmacokinetic characteristics of TRAIL formulas were investigated in rats following intravenous administration. Male Sprague-Dawley rats (average body weight, 200 g) were cannulated in the jugular vein a day before the experiments. Animals were randomly divided to four groups and each group was administered 100 μg kg"1 (protein base) of TRAIL formulas. Blood samples were collected from the cannulated jugular vein at different time points (2, 5, 10, 30, 45 min and 1, 1.5, 2, 4, 6, and 12 h) after intravenous injection, and plasma were obtained by centrifugation and stored at -70°C until required for the assay. Active TRAIL concentrations in rat plasma were measured using commercial TRAIL ELISA
kits (BioSource International, Inc.). Pharmacokinetic parameters were calculated from plasma concentration profiles by using non-compartment model analysis.
In vivo antitumor activity
The antitumor effects of TRAIL formulas were investigated in HCT116 tumor- bearing mice (n=6). Briefly, freshly harvested HCT116 cells (3xl06 cells per mouse) were inoculated subcutaneously into BALB/c athymic mice. Five days later, mice were treated with samples of TRAIL, TRAIL/PEG and TRAIL/4 (150 μg per mouse, i.v.) every 2 days. Saline was treated as a control. Tumor volumes were monitored for 20 days. Tumor volumes were calculated using longitudinal (L) and transverse (W) diameters using V = (L W2) 2"1 and tumor growth inhibition (TGI) percent values were calculated using the formula TGI % = (1 - T C"1) x 100, where T and C are the tumor volume of drug treated and control groups. Immunohistology
Tumor cell apoptosis in vivo was investigated after TRAIL treatment in HCT116 tumor-bearing mice. At 20 day after tumor cell inoculation, tumor tissues were recovered from euthanized animals. Formalin-fixed, paraffin-embedded tissue blocks were sectioned (5 μιη) and apoptotic cell death in tumor tissues was visualized by performing TdT-mediated dUTP nick end labeling (TUNEL) assays using a commercial kit (In Situ Cell Death
Detection Kit, Roche, Mannheim, Germany). For evaluation of kidney tissues, the kidneys were harvested at the end of the experiment, fixed with 10% formalin and blocked by paraffin. Cross-sliced sections of the kidneys were stained with hematoxylin and eosin (H&E) and examined under a light microscopy.
Example 2: Effects of Ni-NTA-PEGs on the stability and bioactivity of TRAIL
Size exclusion chromatography (SEC)
The formation of interaction complexes between TRAIL and Ni-NTA-PEGs were confirmed by size exclusion chromatography (SEC). An active TRAIL including an N- terminal H6 and trimer-forming zipper sequence, H6-ILZ-hTRAIL (114 - 281) (MW 22 kDa), was purified and used as previously reported (S. Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729). As shown in Figure 2A, TRAIL mixed with PEG without the NTA moiety, TRAIL/PEG, did not form any complexes. However, the addition of 1 and 4, producing TRAIL/1 and TRAIL/4, exhibited increasing hydrodynamic radii due to the interaction of TRAIL with the NTA appendage possessing high affinity towards H6. TRAIL/4 showed a similar SEC profile compared to that of covalently bound N-terminal PEGylated TRAIL-
PEG5K without free TRAIL at a feed molar ratio of TRAIL: 4 above 1:5. In contrast, TRAIL/1 failed to reach complete complexation at any ratio.
Binding interaction kinetics
To explore a substantial difference in complexation profiles between TRAIL/1 and TRAIL/4, the interaction kinetics of 1 and 4 with TRAIL was studied by measuring binding constants using BIAcore. The KD value of 1 with TRAIL was 41.6 μιη and 4 was 17.7 μιη (Figure 4). The results demonstrate that the incomplete complexation of TRAIL/1 is probably because of the higher KD value and the induced steric hindrance of 1 in the buffer. After fixing the ratio at 1:5, the stability of each formula was investigated in 20 mM PBS, pH 7.4, at 37 °C, without any stabilizing agents such as Tween 20 and concentrated glycerol and sucrose (S. Frokjaer, D. E. Otzen, Nat. Rev. Drug Discov. 2005, 4, 298-306). Native TRAIL and TRAIL/PEG at a concentration of 400 μg mL"1 (based on the protein concentration) showed rapid aggregation and precipitation, losing more than 70% of the protein in an hour (Figure 2B) because of its low stability and solubility at physiological pH.
In contrast, both 1 and 4 improved stability and reduced precipitation of TRAIL under the same conditions. More than 50% of TRAIL/4 was found to be stable twelve hours after incubation; however, it gradually lost stability.
In vitro biological activity using MTT assays
The bioactivity of each formula was examined based on tumor cell-specific cytotoxicity measured by MTT assays following incubation of TRAIL-based formulas (protein concentration from 10_1-104 ng mL"1) in human colon cancer HCT116 cells (Figure 2C and Table 1). TRAIL and TRAIL/PEG showed a marked apoptotic effect on HCT116 cells. Cytotoxicity of TRAIL associated with 1 and 4 was slightly decreased with increasing NTA affinities but retained 43.7+7.1 and 20.3+1.6% of bioactivity from native TRAIL. The observed IC50 value of TRAIL/4 was similar to that of previously reported TRAIL-PEGSK s (Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729). To confirm TRAIL'S tumor cell specificity, the same TRAIL formulas were treated in normal cells (fibroblast CCD-986sk) and showed no toxicity (Figure 6A). In terms of cytotoxicity of Ni-NTA-PEGs, 1 and 4 were nontoxic both to normal fibroblasts CCD-986sk and HCT116 cells (Figures 6B and 6C). Taken together, in vitro assays demonstrated that simple addition of 1 and 4 to TRAIL was able to provide extended stability in solution and reduced aggregation, while salvaging the bioactivity of TRAIL.
Table 1 : Pharmacokinetic arameters
[a] : the half maximal inhibitory concentration, (ng/mL)
[b] : clearance, (ml/min)
[c] : elimination half- life, (min). *P>0.001 vs. TRAIL alone and TRAIL/PEG.
[d] : TRAIL covalently conjugated with PEG5k
Example 3 : Effects of Ni-NTA-PEGs on the pharmacological efficacy of TRAIL
After validation of TRAIL/Ni-NTA-PEGs in vitro, the PK of TRAIL/1, TRAIL/4 and TRAIL-PEG5K, TRAIL covalently conjugated with the same molecular weight PEG, in rats after intravenous (IV) injection was analyzed. Total active TRAIL plasma levels were measured by enzyme-linked immunosorbent assay (ELISA). All PK parameters are summarized in Table 1 and illustrated in Figure 3A and Figure 6.
Pharmacokinetics of TRAIL, TRAIL/pEG, and TRAIL with Ni-NTA-PEG analogues 1 and 4
It has been reported that TRAIL has a short half-life of 5-10 minutes in rat, mainly through rapid renal clearance (S. K. Kelley, et al., /. Pharmacol. Exp. Ther. 2001, 299, 31- 38). In accordance with reported values, intravenously injected TRAIL and TRAIL/PEG were rapidly eliminated from rats within one hour (Figure 3A). In contrast, TRAIL/4 showed a prolonged elimination half-life and maintained activity up to 6 hours post-injection.
Bioavailability as determined by AUC (area under the curve)
Furthermore, bioavailability of TRAIL/4, as determined by the area under the curve (AUC) analysis, was enhanced by 3.8 + 0.6 and 2.1+0.3-fold compared to that of native TRAIL and TRAIL/1, respectively (Figure 3B). Because 4 showed significantly extended stability in solution and improved PK parameters of TRAIL both in vitro and in vivo compared to 1, 4 was chosen for further PD testing in HCT116-tumor bearing mice.
Antitumor effect of 4 for in vivo applications
To demonstrate the utility of 4 for in vivo applications, the antitumor effect was investigated in tumor models by continually monitoring tumor volumes while treating mice with TRAIL every 5 days. As shown in Figure 3C, all formulas, TRAIL, TRAIL/PEG and TRAIL/4, suppressed tumor growth. However, mean tumor growth with tumor growth inhibition (TGI) values were only maintained by TRAIL/4 throughout the study period (at day 20, TRAIL, TRAIL/PEG and TRAIL/4 had TGI values of 15.1 + 7.6%, 19.2 + 5.3% and 51.8 + 7.1%, respectively), and tumor size rebound was observed in all other formulas. TGI
value was calculated using the formula: [l-(Vr/Vc) xl00%], where VT and Vc are the tumor volume of drug treated and control groups, respectively.
Apoptotic cell death in tumors from mice treated with TRAILs
At the end of the study, tumor tissues were harvested and apoptotic cells in tumor sections were visualized by TdT-mediated dUTP nick end labelling (TUNEL) assays (Figure 3D). TRAIL/4 treated tissues demonstrated increased tumor cell apoptosis compared to those of native TRAIL and the other formulas. In addition, at any injected dose of native TRAIL (50-1000 μg per mouse), TGI of 50% was not achieved under the experimental conditions. Because the major human adverse event related to high PEG exposure is renal toxicity (C. Krejsa, et al., Nat. Rev. Drug Discov 2006, 5, 507-521), acute renal toxicity of 4 was examined by histological investigation of renal tissues after PD studies. No sign of toxicity was observed for any formula (Figure 3D, Right panels). The experimental results from in vitro assays to PK and PD in vivo studies consistently exemplify that an appropriate Ni-NTA-PEG molecule significantly and positively affect the physicochemical properties of His-tagged proteins in vitro and in vivo while maintaining bioactivity. Collectively, TRAIL/4 demonstrated 3- to 4-fold improved efficacy over native TRAIL in terms of solution stability, in vivo half-life and bioavailability. As previously described, covalent TRAIL- PEGSK, retained more than 80% stability in physiological buffer for 24 hours and showed a 10-fold and 30-fold increase in bioavailability compared to TRAIL alone after intraperitoneal (IP) (S. Y. Chae, et al., Mol. Cancer Ther. 2010, 9, 1719-1729) and IV injections (Table 1), respectively. Non-covalently attached Ni-NTA-PEG can be gradually released from the His- tagged protein in the blood.
Surprisingly, however, based on the TGI value, TRAIL/4 demonstrated similar antitumor efficacy to TRAILPEG5K in the same tumor model. This likely due to the different in vivo bioactivities between TRAIL-PEG5K and TRAIL/4; TRAILPEG5K has reduced activity (50% versus TRAIL) for all time points, whereas TRAIL/4 can fully recover its bioactivity once the Ni-NTAPEG is released from it in the blood. Because the administration route (IP versus IV) and dosing profiles are different, the results cannot be directly compared. Note that the technique offers all of the benefits of site-specific
PEGylation without further chemical modification and purification processes. Once 4 is added to protein in solution, the protein can be highly concentrated and freeze-dried.
Because an excess of 4 does not interfere with the bioactivity and PK of the protein, one can easily add the analogues as stabilizers. Moreover, the observed pseudo-PEGylation effect can be achieved by a simple incubation, with the total preparation time less than 30 minutes.
OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
Claims
1. A method of improving in vitro and in vivo pharmacokinetic parameters of a peptide comprising:
coupling a poly-histidine tag (His-tag) to said peptide;
mixing said His-tagged peptide with a complex comprising a metal, nitrilotriacetic acid (NTA) and PEG (poly(ethylene glycol)) or analogue thereof,
thereby producing a His-tagged peptide/ Ni-NTA-PEG composition and improving in vitro and in vivo pharmacokinetic parameters.
2. The method of claim 1, further comprising administering said composition to a subject.
3. The method of claim 1, wherein said pharmacokinetic parameter comprises a prolonged half-life, increased bioavailability, decreased clearance, and reduced aggregation.
4. The method of claim 1, wherein said composition is produced within 30 minutes.
5. A compound, comprising a structure of the following Formula (I):
-ooc
"I
,N . -coo-
NTA represents COO" ;
Ri is optionally substituted alkyl, optionally substituted alkoxy, or hydroxyl;
i is 0 or a positive integer;
L is a linker group;
m is a positive integer; and
M is a metal ion.
6. The compound of claim 5, wherein M is Ni2+.
7. The compound of claim 5, wherein i is 1 or 2.
9. The compound of claim 5, wherein m is 1, 2 or 3.
10. The compound of claim 5 is
11. A composition comprising a complex comprising a metal, nitrilotriacetic acid (NT A) and PEG (poly(ethylene glycol)) or analogue thereof.
12. The composition of claim 11, wherein the metal is selected from the group consisting of Ca2+, Co2+, Cu2+, Ni2+, or Zn2+.
13. The composition of claim 12, wherein the metal is Ni'
14. The composition of claim 11, wherein said composition further comprises a peptide comprising a poly-histidine tag (His-tag).
15. The composition of claim 11, wherein the Ni-NTA-PEG complex is non-covalently bound to the peptide.
16. The composition of claim 11, wherein said Ni-NTA-PEG analogue comprises
17. A kit comprising the composition of claim 1.
18. A kit comprising the composition claim 11.
19. The kit of claim 18, further comprising a His-tagged peptide.
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