WO2014054046A1 - Fibrilles, agrégats et multimères réversibles ordonnés de protéines et leurs utilisations - Google Patents

Fibrilles, agrégats et multimères réversibles ordonnés de protéines et leurs utilisations Download PDF

Info

Publication number
WO2014054046A1
WO2014054046A1 PCT/IL2013/050813 IL2013050813W WO2014054046A1 WO 2014054046 A1 WO2014054046 A1 WO 2014054046A1 IL 2013050813 W IL2013050813 W IL 2013050813W WO 2014054046 A1 WO2014054046 A1 WO 2014054046A1
Authority
WO
WIPO (PCT)
Prior art keywords
reversible
aggregate
ordered
protein
fibril
Prior art date
Application number
PCT/IL2013/050813
Other languages
English (en)
Inventor
Yaron Dekel
Jacob Pitcovski
Original Assignee
Gavish-Galilee Bio Applications Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gavish-Galilee Bio Applications Ltd filed Critical Gavish-Galilee Bio Applications Ltd
Publication of WO2014054046A1 publication Critical patent/WO2014054046A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates in general to ordered reversible fibrils, aggregates and multimers of therapeutic proteins for use as depot reservoir, sustained release formulation and stabilizer.
  • a crucial aspect of native protein formulations is stability. Proteins, in their native conformation, tend, in time, to misfold and aggregate, reaching a more favorable thermodynamic state. This state bears lower energy levels and thus is unavoidable. In the form of amorphous aggregates, proteins lose their native conformation, sediment and as a consequence, loose their biological activity. The process is irreversible and detrimental (Gazit, 2002). In order to overcome this problem, various formulations were developed; some of them contain toxic irritant materials with possible carcinogenic effects (Pfohler et al., 2008, Rajpar et al., 2006). For example, the association between insulin analogues and cancer is widely studied in vitro and epidemiological ⁇ , although the topic is still controversial.
  • Antibodies and antibody-derived molecules are fast growing classes of biopharmaceutical products.
  • a recent study on the nature of IgG aggregation suggests that IgG soluble aggregates are composed of monomers, in which at least some fraction of the protein chain has adopted an amyloid-like structure, similar to that of single-domain proteins such as insulin (Brummitt et al., 2011a,b). Indeed, little is known about self-assembly of high molecular weight proteins.
  • a report of a study of high molecular weight proteins of about 50KD demonstrated the ability to form fibrils in vitro. These fibrils were poorly stained with common fibrillation dyes like Congo red or Thioflavin-T (ThT) (although significantly), therefore leading to the assumption that these fibrils are mostly non-amyloidogenic (Ramshini et al., 2011).
  • Protein fibrils were used in an attempt to deliver insulin orally (Dekel et al., 2010; WO 2009/108554). Insulin, a major therapeutic peptide worldwide was driven to its fibrillar state inside different micro particles. These formulations proved to be resistant in various stimulating conditions that mimic the gastro-intestinal tract. Moreover, the fibrils formulations were shown to be therapeutically active in vivo (Dekel et al., 2010; WO 2009/108554). Thus, protein fibrillation can be viewed positively and not only detrimentally.
  • Growth hormone also known as somatotropin or somatropin, is a 191 -amino acid, single-chain peptide hormone that stimulates growth, cell reproduction and regeneration in humans and other animals. It is used as a prescription drug in medicine to treat children's growth disorders and adult growth hormone deficiency. It is also used in dairy cows to increase milk production, but due to regulations it is not used in raising cattle for beef or for raising poultry. Current regimen for the treatment of humans with growth hormone dictates daily injection which is associated with much discomfort.
  • Herceptin an important human 150kD monoclonal IgG, first line treatment in HER2+ breast cancer, can be driven to a self-assembled polymeric state at pH 2 and 37°C under gentle agitation. It was further found that the antibody was present in the aggregate in an intact state and that changing the thermodynamic state of the solution of the self-assembled structures from aggregation-favoring conditions (high protein concentration, pH 2) to non-aggregation- favoring conditions (low protein concentration, physiologic pH), by diluting the self- assembled structure solution in a physiological solution, resulted in release of active antibodies from the self-assembled structures.
  • the present invention provides a reversible ordered fibril, aggregate or multimer of a therapeutic protein, wherein the therapeutic protein is selected from (i) a high molecular weight protein; or (ii) a growth hormone, and the therapeutic protein is active in soluble form.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a reversible ordered fibril, aggregate or multimer of a therapeutic protein, wherein the therapeutic protein is selected from (i) a high molecular weight protein; or (ii) a growth hormone, and the therapeutic protein is active in soluble form.
  • the present invention provides a method for preparing a reversible ordered fibril, aggregate or multimer of a therapeutic protein, wherein the therapeutic protein is selected from (i) a high molecular weight protein; or (ii) a growth hormone, and the therapeutic protein is active in soluble form, the method comprising solubilizing the therapeutic protein in an aqueous solution having a pH of between about 1.5 to about 4, at a temperature of between about 25°C to about 50°C, during gentle agitation for a period of between about 6 hours to about 30 days.
  • the present invention provides a method for treatment of a disease, disorder or condition selected from the group consisting of breast cancer, chronic lymphocytic leukemia, transplant rejection, systemic lupus erythematosus, colorectal cancer, age related macular degeneration, anaplastic large cell lymphoma (ALCL), Hodgkin's lymphoma, cryopyrin-associated periodic syndrome (CAPS), head and neck cancer, Crohn's disease, postmenopausal osteoporosis, solid tumor s bony metastases, paroxysmal nocturnal hemoglobinuria, psoriasis, acute myelogenous leukemia, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, non-Hodgkin's lymphoma, melanoma, multiple sclerosis, allergy-related asthma, respiratory syncytial virus infection, growth hormone deficiency in children or in adults,
  • Figs. 1A-C show ThT fluorescence (4-500 ⁇ ) peak values of (A) Herceptin (lmg/ml), (B) Herceptin (5mg/ml) and (C) IgY (lmg/ml) during 3 hours of self-assembly process. Different ThT concentrations symbols are given in legend.
  • Fig. 2 ThT fluorescence ( ⁇ ) peak values of Herceptin (lmg/ml) during 3 days of self-assembly. Solid line -Assembly in 370C with gentle agitation. Dashed line - Assembly in 37°C with stirring.
  • Figs. 3A-B show ThT fluorescence ( ⁇ ) for (A) Herceptin (lmg/ml) after 20 hours into self-assembly process and (B) IgY (lmg/ml).
  • Figs. 4A-B show (A) CD spectroscopy of native (dashed), self-assembled (solide) Herceptin. (B) CD spectroscopy of native (solid), fibrillar (dashed) human Insulin.
  • Figs. 5A-B shows gel images of native and self-aggregated Herceptin under (A) reduced conditions; (B) - non-reducing conditions.
  • Figs. 6A-G show atomic force microscopy (AFM) images of different Herceptins formulaions.
  • A native Herceptin with height graph in nm.
  • B monomeric and polymeric Herceptin.
  • C and
  • D different polymeric Herceptin structures.
  • Figs. E-G show different polymeric Herceptin sizes with height graph.
  • Figs. 7A-C show activity restoration of assembled proteins.
  • B decrease in fluorescence of native FITC-Herceptin after competition with assembled (left bar) or native (right bar) Herceptins.
  • C binding activity of centrifuged Herceptin preparation divided into pellet (left bar) and supernatant (right bar).
  • Fig. 8 depicts multimeric IgY binding to VP2 antigen.
  • X-axis time period of disassociation. disassociation
  • FIGs. 9A-B show binding ability of control freshly prepared native Herceptin.
  • A image of Herceptin suspensions vials, native (left) and assembled (right) after 3 days of agitation in 37°C and 40 days at room temperature.
  • B Antibodies from (A) were tested for activity after 160 days at room temperature.
  • Herceptin polymeric structures (or native antibodies) were diluted prior to experiment 1: 100 and up to 3 days dilution.
  • Fig. 10 shows maternal titer of anti-VP2 antibody 18 hours, 1 week and 2 weeks after treatment.
  • the present invention is directed to a reversible ordered fibril, aggregate or multimer of a therapeutic protein, wherein the therapeutic protein is selected from (i) a high molecular weight protein; or (ii) a growth hormone, and the therapeutic protein is active in soluble form, i.e. the individual polypeptides that dissociate from the reversible ordered fibril, aggregate or multimer are active.
  • the therapeutic protein is selected from (i) a high molecular weight protein; or (ii) a growth hormone, and the therapeutic protein is active in soluble form, i.e. the individual polypeptides that dissociate from the reversible ordered fibril, aggregate or multimer are active.
  • reversible is used interchangeably herein with the term “reversibly water insoluble” and refers to the feature of the ordered fibril, aggregate or multimer of being able to transform from an insoluble ordered fibril, aggregate or multimer to a water soluble ordered fibril, aggregate or multimer.
  • the term also refers to the capability of the protein of transforming from its conformation in a fibril, aggregate or multimer to a conformation of a soluble native protein.
  • aggregate and multimer of a therapeutic protein refer to the joining together of discrete native proteins in an ordered, non-covalently and reversible manner. Each protein in the multimer retains its fundamental native structure.
  • fibrous structure of a therapeutic protein as used herein refers to the joining together of non-native proteins that expose their hydrophobic residues to the surface and form fibrous structure with distinctive beta sheet conformation in a diameter of 10-15nm.
  • active protein or “protein [that] is active” as used herein to describe the function of an antibody that has been manipulated, for example to undergo aggregation, refers to such an antibody that exhibits the binding affinity and antigen- specificity of an identical antibody that has not been manipulated (other than being dissolved in a solution).
  • active protein or protein [that] is active as used herein to describe the function of an enzyme refers to such an enzyme that exhibits the affinity, specificity and kinetic features of an identical enzyme that has not been manipulated (other than being dissolved in a solution).
  • active protein or "protein [that] is active” as used herein to describe the function of a ligand to a receptor, such as growth hormone, refers to such a ligand that exhibits the affinity, specificity and downstream signaling of an identical ligand that has not been manipulated (other than being dissolved in a solution).
  • the high molecular weight protein has a molecular weight of at least about 50kDa, for example between about 50kDa and about 200kDa.
  • the molecular weight of the high molecular weight protein may be between 50kDa and 200kDa, 55kDa and 200kDa, 60kDa and 200kDa, 65kDa and 200kDa, 70kDa and 200kDa, 75kDa and 200kDa, 80kDa and 200kDa, 85kDa and 200kDa, 90kDa and 200kDa, 95kDa and 200kDa, lOOkDa and 200kDa, HOkDa and 200kDa, 120kDa and 200kDa, 130kDa and
  • the high molecular weight protein has a molecular weight of about 150kDa.
  • the reversible ordered fibrils, aggregates or multimers of the present invention dissociate in non-aggregating conditions and form an active soluble form of the therapeutic protein.
  • the non-aggregating conditions may be obtained by, but is not limited to, dilution of the reversible ordered fibrils, aggregates or multimers in aqueous solutions such as water, different physiological buffers (such as phosphate-buffered saline (PBS) or tris(hydroxymethyl)aminomethane (TRIS) saline), acidic or alkali buffers, blood, gastric or intestinal fluids (simulated or endogenous), human or animal serum; buffers containing different proteins such as bovine or human serum albumin or protamine sulfate in concentrations from about O. lmg/ml to about lOmg/ml.
  • PBS phosphate-buffered saline
  • TIS tris(hydroxymethyl)aminomethane
  • the therapeutic protein is selected from the group consisting of a monomeric protein, a homooligomeric protein (i.e. a protein that consists of several identical polypeptides and has a tertiary structure) and a heterooligomeric protein (i.e. a protein that consists of several different polypeptides and has a tertiary structure).
  • a monomeric protein e.g. a protein that consists of several identical polypeptides and has a tertiary structure
  • a heterooligomeric protein i.e. a protein that consists of several different polypeptides and has a tertiary structure.
  • the high molecular weight protein is selected from the group consisting of an antibody, a-iduronidase, a-galactosidase, arylsulfatase B, tissue plasminogen activator, glucocerebrosidase and infectious bursal disease virus VP2 subunit (SEQ ID NO: 1), and in particular an antibody selected from the group consisting of IgG, IgA, IgM and IgY.
  • the antibody is an IgG monoclonal antibody.
  • the IgG antibody is a human, chimeric or humanized monoclonal antibody.
  • the monoclonal antibody binds to an antigen associated with a disease selected from the group consisting of breast cancer, chronic lymphocytic leukemia, transplant rejection, systemic lupus erythematosus, colorectal cancer, age related macular degeneration, anaplastic large cell lymphoma (ALCL), Hodgkin's lymphoma, cryopyrin-associated periodic syndrome (CAPS), head and neck cancer, Crohn's disease, postmenopausal osteoporosis, solid tumor s bony metastases, paroxysmal nocturnal hemoglobinuria, psoriasis, acute myelogenous leukemia, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,
  • a disease selected from the group consisting of breast cancer, chronic lymphoc
  • the antibody is selected from the group consisting of Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Belimumab, Bevacizumab, Brentuximab vedotin, Canakinumab, Cetuximab, Certolizumab pegol, Daclizumab, Denosumab, Eculizumab, Efalizumab, Gemtuzumab, Golimumab, Ibritumomab, tiuxetan, Infliximab, Ipilimumab (MDX-101), Muromonab-CD3, Natalizumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tocilizumab, Tositumomab and Trastuzumab.
  • this list recites the scientific names of the antibodies and
  • the present invention provides a reversible ordered multimer of an IgG antibody, wherein the IgG antibody is active in soluble form.
  • the IgG antibody is Trastuzumab.
  • the fibrils, aggregates or multimers are formed at pH 2 under gentle agitation.
  • the term "gentle agitation” as used herein refers to agitation of the solution by swirling it on a shaker at a rate of between about 30 revolutions per minutes (RPM) to about 90 RPM.
  • the antibodies in the ordered state form multimers in the shape of spheres that join together and form dimers, tetramers and bigger structures of up to 300nm in length and 16nm in height.
  • the reversible ordered fibrils, aggregates or multimers form spherical structures ordered as monomeric spheres of the length of about lOnm and height of about 2-3 nm or oligomeric spheres of the length of up to about 500 and about 20nm in height.
  • the present invention contemplates the formation of reversible ordered fibrils, aggregates or multimers of growth hormone, as shown in Example 7. These fibrils are unencapsulated and uncoated, i.e. no other polymer or other material is comprised in or around the fibril. Based on the evidence collected from the work on antibodies disclosed herein below, it is expected that the growth hormone fibrils will dissolve in a physiological solution and that the re-dissolved growth hormone is active, i.e. capable of binding to and activating its native receptor (growth hormone receptor) both in vitro and in vivo.
  • the dissolution of the growth hormone fibril will be extended over time, as has been shown herein for antibody fibrils (Example 5) and that the fibril will function as a reservoir for sustained release of growth hormone.
  • the fibrils will therefore provide for an administration regimen at a frequency that is lower than once a day, e.g. once a week. It is possible that the fibrils are suitable for oral administration.
  • the present invention further provides a reversible ordered fibril, aggregate or multimer growth hormone.
  • the growth hormone is human growth hormone (SEQ ID NO: 2).
  • the reversible ordered fibril, aggregate or multimer of the present invention acts as a stabilizer and is for prolonging the shelf life of the therapeutic protein comprised therein as compared with the shelf-life of the corresponding therapeutic protein in solution.
  • the reversible ordered fibril, aggregate or multimer of the present invention provides sustained release of the therapeutic protein forming the ordered fibril, aggregate or multimer.
  • the reversible ordered fibril, aggregate or multimer of the present invention is a unencapsulated and uncoated fibril, aggregate or multimer, e.g. the reversible ordered fibril, aggregate or multimer is not encapsulated in a lipidated glycosaminoglycan particle.
  • the present invention is directed to a reversible ordered fibril, aggregate or multimer of a therapeutic protein for use in treatment of a disease, disorder or condition selected from the group consisting of breast cancer, chronic lymphocytic leukemia, transplant rejection, systemic lupus erythematosus, colorectal cancer, age related macular degeneration, anaplastic large cell lymphoma (ALCL), Hodgkin's lymphoma, cryopyrin- associated periodic syndrome (CAPS), head and neck cancer, Crohn's disease, postmenopausal osteoporosis, solid tumor s bony metastases, paroxysmal nocturnal hemoglobinuria, psoriasis, acute myelogenous leukemia, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, non-Hodgkin's lymphoma, melanoma, multiple sclerosis, allergy-related asthma, respiratory s
  • the invention also contemplates the use of fibrils and ordered reversible aggregates of antibodies, for example bovine or horse IgG or chicken IgY, for toxin neutralization, treatment for diphtheria, tetanus, botulism and snake envenomation or treatment or prevention of disease in other mammals, such as domesticated birds, sheep, goats and cattle.
  • antibodies for example bovine or horse IgG or chicken IgY, for toxin neutralization, treatment for diphtheria, tetanus, botulism and snake envenomation or treatment or prevention of disease in other mammals, such as domesticated birds, sheep, goats and cattle.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and reversible ordered fibril, aggregate or multimer of a therapeutic protein as defined herein above.
  • the therapeutic protein of the pharmaceutical composition is an antibody of type IgG; in other embodiments the therapeutic protein is human growth hormone.
  • the ordered reversible fibrils, aggregates and multimers are very stable at room temperature and that the conditions used to form the fibrils, aggregates or multimers may be tailor-made to dissociate faster or slower.
  • the inventors found that the dissociation product of an antibody fibril is active antibodies that can be released in a sustained release manner from the polymer while maintaining their activity and affinity throughout the process. As further shown in Example 5, the ability to release antibodies is dependent on the dilution matrix and the polymeric status and might also influence long term stability and enable a greater shelf life.
  • the polymeric structure of the high molecular weight protein such as Herceptin, is unique in that it serves as the depot reservoir, a releasing matter of active antibodies and as a stabilizer for the antibodies within it.
  • These three important entities provide this formulation the promising characteristics needed for e.g. oral delivery administration protocols not only for antibodies, but for any therapeutic high molecular weight protein, as for example growth hormone (see below).
  • the enhanced shelf-life itself is an important improvement over existing formulations of therapeutic proteins; for example, a therapeutic antibody that is prescribed for administration by injection may be stored for prolonged time periods at room temperature and when needed, is diluted in an appropriate physiological solution suitable for injection. When the ordered reversible fibrils, aggregate or multimer is completely dissolved, the solution is injected in the patient according to existing approved protocols.
  • the pharmaceutical composition is adapted for oral administration and/or it is formulated for sustained release of the therapeutic protein.
  • the present invention is directed to a method for preparing a reversible ordered fibril, aggregate or multimer of a therapeutic protein as defined herein, comprising solubilizing the therapeutic protein in an aqueous solution, such as water, saline or physiological buffer, having a pH of between about 1.5 to about 4, at a temperature of between about 25°C to about 50°C, during gentle agitation.
  • an aqueous solution such as water, saline or physiological buffer, having a pH of between about 1.5 to about 4, at a temperature of between about 25°C to about 50°C, during gentle agitation.
  • the protein is solubilized at pH 2. In other embodiments, the protein is solubilized at 37°C.
  • the method comprises adding to the solution of dissolved therapeutic protein a particle selected from a Teflon (poly(l,l,2,2-tetrafluoroethylene) particle, a N-isopropylacrylamide-co-N-tert-butylacrylamide (NIPAM/BAM) copolymer particle or a glass particle, wherein said particle increases the formation of hydrophobic surfaces in the solution.
  • a particle selected from a Teflon (poly(l,l,2,2-tetrafluoroethylene) particle, a N-isopropylacrylamide-co-N-tert-butylacrylamide (NIPAM/BAM) copolymer particle or a glass particle, wherein said particle increases the formation of hydrophobic surfaces in the solution.
  • the Teflon particle may have a diameter of 100-300 nm; the NIPAM/BAM copolymer particle may comprise 50% NIP AM and 50% BAM or 90% NIP AM and 10% BAM, and the NIPAM/BAM copolymer particle may have a diameter of 70nm to 200 nm; and said glass particle may have a diameter of 100-300 nm.
  • the Teflon particle has a diameter of 200 nm. In certain embodiments, the glass particle has a diameter of 200 nm.
  • treating refers to means of obtaining a desired physiological effect.
  • the effect may be therapeutic in terms of partially or completely curing a disease and/or symptoms attributed to the disease.
  • the term refers to inhibiting the disease, i.e. arresting its development; or ameliorating the disease, i.e. causing regression of the disease.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local. In certain embodiments, the pharmaceutical composition is adapted for oral administration.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered.
  • the carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.
  • a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate
  • a disintegrating agent such as alginic acid, maize starch and the like
  • a lubricant or surfactant such as
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives e.g
  • compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.
  • compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • PBS phosphate-buffered saline
  • ThT Thint
  • ThT Sigma Chemical Co. St. Louis, USA, Ex.450nm, Em.460-600nm, peak at 482nm
  • ThT concentration of ThT and aliquots of ⁇ from self-assembled suspensions were mixed together and measured in a quartz cuvette in Varian- Cary eclipse fluoremeter.
  • Recombinant human insulin Biological Industries, Israel was used in parallel as a control peptide.
  • Self-assembled samples were tested every 30 or 60 minutes for the first 8 hours and then daily up to seven days.
  • B - Self-assembled structures were visualized by AFM.
  • SK-BR-3 human breast cancer cell line expressing HER2
  • FCS fetal calf serum
  • penicillin streptomycin solution Biological Industries, Beit Haemek, Israel
  • FACS Fluorescence-activated cell sorting
  • SKBR3 cells were trypsinized, washed (as in 2.7) and divided into different tubes (300,000cells/tube). Cells were incubated for 1 hour in 4°C, gentle agitation (30-90 rpm) with the following solutions: 1. ⁇ . PBS PLUS ⁇ NHS-FITC (Pierce Biotechnology , Rockford, 1L , USA) conjugated to Herceptin (lmg/ml). The conjugation procedure was according to manufacture protocol. Briefly: lmg of NHS- Fluorescein was reconstitute with ⁇ DMSO. NHS -Fluorescein was mixed with antibody solution, 15: 1 molar excess, and incubated for 2 hours in room temperature.
  • Stability assay Self-assembled Herceptin polymers (lmg/ml) were prepared for 30 days under gentle agitation (30-90 rpm) at 37°C. Native Herceptin was kept in the same conditions for control. Vials of self-assembled and native Herceptin were stored at room temperature without agitation and assayed for activity after extra 120 days. In a different experiment, the same preparations as above were kept for 2 months in 60°C with gentle agitation.
  • Example 1 Determinations of conditions for Herceptin self-assembly.
  • ThT fluorescence at 482nm is used to detect growing protein fibrils in solution.
  • ThT fluorescence intensities for both preparations are similar over time (shown only for IgG for brevity).
  • the rate of fibril or multimer formation can be controlled by varying the ionic strength of the solution, e.g. from water to regular PBS (lxPBS) and up to PBS concentrated 10 times the regular PBS (lOxPBS). The higher the ionic strength, the faster the procedure reaches its endpoint.
  • Fig. 5A is a gel image with reducing conditions (DTT added) in which both preparations, native and self-assembled, locate according to the sizes of the heavy (50,000Da) and light (25,000) subunits.
  • Fig. 5B is a gel image the same samples as in Fig. 5A without reducing conditions in which both self-assembled and native antibodies bands remain in their complete size (150,000Da) position with no apparent differences between them.
  • Self-assembled and native Insulin were examined in reducing conditions with similar results (not shown).
  • beta-Lactoglobulin It has been reported for beta-Lactoglobulin that it can form self-assembled structures during heating to 80°C but when subjected to gel electrophoresis, the self- assembled structures dissociate into different peptides as the main beta-Lactoglobulin band becomes weaker and smaller bands appear (Oboroceanu et al., 2010). The authors concluded that during the formation of self-assembled structures, the beta-Lactoglobulin dissociated into smaller peptides that form self-assembled structures. According to the results presented here, no significant dissociation to other peptides species is apparent; this might be due to the preparation procedure here in which the self-assembled structures are made in physiological temperature.
  • FIG. 6A demonstrates a representative example of native Herceptin antibodies.
  • samples were diluted 8000 fold (and not 1000 fold as in the case of the self-assembled preparations) as discrete monomers were not visible due to smear (abundance of material).
  • an apparent tri-nodular shape, representative of the antibody structure (Fc and Fab) is evident and the molecule height (2-3nm) and length (lOnm).
  • FIG. 6 C, D and E are representative examples of the self-assembled structures and the monomers composing them.
  • the structures seen are polymers composed of discrete antibodies as their building block.
  • Fig. 6C discrete monomers are evident yet their shape does not resemble the common antibody tri-nodular shape but rather a rounded sphere (compare Figs. 6A and 6C).
  • Figs. 6D, E These rounded monomers are assembled to form polymers that in many cases start from 'strings' of rounded antibodies that are folded to form larger globular structures.
  • the assembled structure representative sizes are given in Figs. 6F, G and H. While monomers retain their regular dimensions (Fig. 6D), there are structures in the height of 6nm (three layers of antibodies) and 30-40 nm in length (Fig. 5G) and larger structures, reaching 15nm in height (6-7 layers of antibodies) and reaching 300nm in length (Fig. 6G).
  • ELISA plates were coated with dissociated VP2 at different dilutions or with native VP2 for control in the same concentrations/dilutions.
  • ELISA plates were coated only with native VP2.
  • VP2 was adsorbed to the ELISA plates in carbonate coating buffer overnight at 4 °C. After a blocking step with skim milk, the plate was incubated with chicken anti-VP2 antibody for 1 h at 37 °C, followed by incubation with rabbit anti- chicken antibody (Sigma) conjugated with horse radish peroxidase (HRP) enzyme for an additional 1 h at 37 °C.
  • HRP horse radish peroxidase
  • VP2 protein was determined by measuring OD at 450 nm as described above. A high absorption was an indication of large amounts of VP2 adsorbed on the ELISA plate.
  • FCS fetal calf serum
  • the actual dilution time is around 1.5h as the incubation time of primary antibody is 1 hour (adding more time for washes, see material and methods for details).
  • the common VP2 vaccination procedure resulted in similar titers to the other two controls might indicate that the amount of the multimeric VP2 that dissociated and entered the blood stream as monomers is at least significantly greater than 5% as the initial amount was lmg/broiler.
  • Example 7 Self-assembly of growth hormone and restoration of its fibrils.
  • Samples are acidified to pH 2 and pH 3 with HC1 (at 37 C, gentle agitation (30-90 rpm).
  • Protocol for ThT analysis in confocoal microscope Suspensions of protein assemblies or control are pipetted (100 ⁇ ⁇ aliquots) into 96-well plates and allowed to dry. Two hundred microliters of a 1% aqueous Thioflavin-S, Sigma Chemical Co. (St.Louis, USA) are added to each well. Three minutes later the Th-S is aspirated and each well is subjected to 3 successive washes with 200 ⁇ water. The wells are then incubated for 20 min with 200 ⁇ of 1% acetic acid. At the end of incubation, the acetic acid is aspirated and each well was washed with water as above, and left to dry.
  • the plate is viewed under a confocal microscope, at excitation and emission wavelengths of 450 nm and 480 nm, respectively [D. Oboroceanu, L. Wang, A. Brodkorb, E. Magner, M.A. Auty, Characterization of beta-lactoglobulin fibrillar assembly using atomic force microscopy, polyacrylamide gel electrophoresis, and in situ fourier transform infrared spectroscopy, J Agric Food Chem, 58 (2010) 3667-3673; N.H. Thomson, The substructure of immunoglobulin G resolved to 25 kDa using amplitude modulation AFM in air, Ultramicroscopy, 105 (2005) 103-110].
  • Example 8 Self-assembly of proteins in the presence of nanoparticles.
  • the protein solution is subjected to gel filtration before it incubation at 37°C with or without 0.01 mg/ml copolymer nanoparticles such as a Teflon (poly(l,l,2,2- tetrafluoroethylene) particle, a N-isopropylacrylamide-co-N-tert-butylacrylamide (NIP AM/BAM) copolymer particle or a glass particle.
  • copolymer nanoparticles such as a Teflon (poly(l,l,2,2- tetrafluoroethylene) particle, a N-isopropylacrylamide-co-N-tert-butylacrylamide (NIP AM/BAM) copolymer particle or a glass particle.
  • NIP AM/BAM N-isopropylacrylamide-co-N-tert-butylacrylamide
  • Nonnative aggregation of an IgGl antibody in acidic conditions part 2: nucleation and growth kinetics with competing growth mechanisms, J Pharm Sci, 100 2104-2119.
  • Gazit E. The "Correctly Folded” state of proteins: is it a metastable state?, Angew Chem Int Ed Engl, 41 (2002) 257-259.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Endocrinology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une fibrille, un agrégat ou un multimère ordonné réversible d'une protéine thérapeutique, ladite protéine thérapeutique étant choisie parmi (i) une protéine à masse moléculaire élevée; ou (ii) une hormone de croissance, et ladite protéine thérapeutique étant active sous forme soluble.
PCT/IL2013/050813 2012-10-04 2013-10-03 Fibrilles, agrégats et multimères réversibles ordonnés de protéines et leurs utilisations WO2014054046A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261709416P 2012-10-04 2012-10-04
US61/709,416 2012-10-04

Publications (1)

Publication Number Publication Date
WO2014054046A1 true WO2014054046A1 (fr) 2014-04-10

Family

ID=50434433

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2013/050813 WO2014054046A1 (fr) 2012-10-04 2013-10-03 Fibrilles, agrégats et multimères réversibles ordonnés de protéines et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2014054046A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108034713A (zh) * 2017-12-29 2018-05-15 北京泱深生物信息技术有限公司 绝经后骨质疏松症诊疗靶点及其应用
CN114894911A (zh) * 2022-03-18 2022-08-12 辽宁成大生物股份有限公司 一种控制牛血清产品质量方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009108554A2 (fr) * 2008-02-27 2009-09-03 Ramot At Tel Aviv University Ltd. Système d’administration de protéine sous forme de fibrilles ou d’agrégats insolubles
US20090258818A1 (en) * 2008-04-07 2009-10-15 National Institute Of Immunology Compositions For the Treatment of Diabetes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009108554A2 (fr) * 2008-02-27 2009-09-03 Ramot At Tel Aviv University Ltd. Système d’administration de protéine sous forme de fibrilles ou d’agrégats insolubles
US20090258818A1 (en) * 2008-04-07 2009-10-15 National Institute Of Immunology Compositions For the Treatment of Diabetes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KIM H.K ET AL.: "Microencapsulation of dissociable human growth hormone aggregates within poly(d,l-lactic-co-glycolic acid) microparticles for sustained release Volume", vol. 229, no. ISSUE, 28 October 2001 (2001-10-28), pages 107 - 116, Retrieved from the Internet <URL:http://dx.doi.org/10.1016/S0378-5173(01)00852-3> *
WANG W. ET AL.: "Protein aggregation-Pathways and influencing factors", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 390, no. ISSUE, 10 May 2010 (2010-05-10), pages 89 - 99, Retrieved from the Internet <URL:http://dx.doi.org/10.1016/j.ijpharm.2010.02.025> *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108034713A (zh) * 2017-12-29 2018-05-15 北京泱深生物信息技术有限公司 绝经后骨质疏松症诊疗靶点及其应用
CN114894911A (zh) * 2022-03-18 2022-08-12 辽宁成大生物股份有限公司 一种控制牛血清产品质量方法
CN114894911B (zh) * 2022-03-18 2023-10-24 辽宁成大生物股份有限公司 一种控制牛血清产品质量方法

Similar Documents

Publication Publication Date Title
US10406212B2 (en) Fibronectin based scaffold domain proteins that bind to myostatin
CN103154036B (zh) 天然人蛋白片段的融合蛋白以产生有序多聚化免疫球蛋白fc组合物
JP6174727B2 (ja) 治療的特性を有するβ１〜４２特異的モノクローナル抗体
JP6283517B2 (ja) 抗体調製物
TWI516500B (zh) 抗β類澱粉蛋白單株抗體
JP2009519915A (ja) 治療ワクチン
JP2004524322A (ja) 血漿増量剤として用いる組換えゼラチン様タンパク質
CN101325972A (zh) 具有治疗性质的Aβ1-42特异性单克隆抗体
WO2014054046A1 (fr) Fibrilles, agrégats et multimères réversibles ordonnés de protéines et leurs utilisations
KR20150082328A (ko) 이중특이성 t-세포 연계체(bites)의 제제
JP2015524418A (ja) 血管内皮細胞への髄膜炎菌の線毛媒介接着の受容体としてのｃｄ１４７
WO2013177116A1 (fr) Modification enzymatique d&#39;un auto-anticorps anti-aqp4 pour la modulation de la maladie de devic
CN108472382A (zh) 抗-因子d抗体变体缀合物及其用途
JP4331798B2 (ja) アレルゲン処方
US20210403897A1 (en) Chemical engineering of erythrocytes to display polypeptides and other biomacromolecules
WO2013045469A1 (fr) Composition d&#39;igy destinée à être utilisée contre la maladie cœliaque
CN113049832A (zh) 定量检测牛乳中过敏原α-乳白蛋白的双抗体夹心法
WO2016058237A1 (fr) Procédé de purification et de préparation de sérum positif d&#39;allergène
EP3644061A1 (fr) Diagnostic des maladies auto-immunes bulleuses
Asaia et al. Injectable Elastin-Like Polypeptide Hydrogel Depot System for Assessment of the Immune-Response-Inducing Efficacy of Sustained Antigen Release Alone
TWI676635B (zh) α－突觸核蛋白Ｃ端胜肽免疫原及其用於治療突觸核蛋白疾病的配方
WO2021220216A1 (fr) Méthodes pour éliminer une hémorragie vitreuse
JP2024514196A (ja) 改変型タンパク質分子を含む機能化された生体膜、並びにその作成方法及び使用
WO2023060151A1 (fr) Épitope d&#39;allergie à l&#39;arachide
Chen A study of the IgM interaction with complement using mouse IgM/IgG2b domain-switched hybrids

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13843896

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13843896

Country of ref document: EP

Kind code of ref document: A1