Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/08—Clostridium, e.g. Clostridium tetani
• • 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
  • A61K47/54—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 compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • 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
  • A61K47/54—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 compound
  • A61K47/555—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 compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
  • A61K47/557—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 compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/74—Inducing cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3513—Protein; Peptide

Definitions

• Antibodies have proven to be an efficacious drug modality for its easy generation, specificity and bio-durability relative to other types of drugs such as small molecule drugs.
• Current antibody therapy can only target extracellular molecules.
• numerous important targets for disease treatment and disease diagnosis are intracellular.
• a number of transcriptional factors, such as STAT3 are among the most crucial yet challenging targets for cancer therapy.
• Provided herein are solutions for these and other needs in the art.
• a cell-penetrating conjugate includes (i) a non-cell penetrating protein, (ii) a phosphorothioate nucleic acid and (iii) a non- covalent linker attaching the phosphorothioate nucleic acid to the non-cell penetrating protein.
• the non-covalent linker includes a biotin-binding domain non-covalently attached to a biotin domain and the phosphorothioate nucleic acid enhances intracellular delivery of the non-cell penetrating protein.
• kit including the cell penetrating conjugate provided herein including embodiments thereof or the pharmaceutical composition as provided herein including embodiments thereof and instructions for use are provided.
• a method of delivering a non-cell penetrating protein into a cell includes contacting the cell with the cell penetrating conjugate as provided herein including embodiments thereof.
• a method of treating a disease in a subject in need thereof includes administering to a subject an effective amount of the cell penetrating conjugate as provided herein including embodiments thereof, thereby threating the disease in the subject.
• FIG. 1 Conjugation scheme: Avidin to antibody/peptide and biotin-oligo to avidin. (1) Avidinylation of IgG; (2) add biotinylated delivery entity (i.e. PS-oligomers). Instead of avidin, streptavidin (or avidin derivatives) may be used; streptavidin provides the opportunity to accept 4 biotins, while avidin accepts just 1 biotin. PS, phosphothioate
• FIG. 4 A and FIG. 4B Direct comparison of attachment of phosphorothioated DNA- oligo vs PEGylation to antibody protein via biotin-avidin/streptavidin for cell penetration and intracellular target recognition. Non-covalent linkage of DNA-oligo's via avidin-biotin to antibody protein is superior to antibody PEGylation.
• Human glioma U251 cells were incubated with anti-STAT3 antibodies (rabbit) modified as indicated for 1 h at 10 mg/ml.
• FIG. 4A Single cell suspensions were prepared and rabbit IgG was assessed by intracellular staining procedure analyzed by flow cytometry.
• FIG. 4A Single cell suspensions were prepared and rabbit IgG was assessed by intracellular staining procedure analyzed by flow cytometry.
• FIG. 6 Modified anti-STAT3 antibodies conjugated to PS-DNA oligos via
• FIG. 10 Tumor growth of mouse B16 melanoma upon treatment with modified anti-T- Bet antibody. 1 ⁇ 105 B16 tumor cells were injected subcutaneously on day 0 in C57/BL6 mice, followed by local administrations of vehicle (PBS), modified control (IgG) and T-bet antibodies (lOug/dose) starting from day 7. Antibody treatments were given every other day. SD shown; T- test: ***) PO.001.
• Nucleic acid or "oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together.
• Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, or complements thereof.
• polynucleotide refers to a linear sequence of nucleotides.
• nucleotide typically refers to a single unit of a
• Phosphorothioate oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Phosphorothioate nucleic acids may also be longer in lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. As described above, in certain embodiments, the phosphorothioate nucleic acids herein contain one or more phosphodiester bonds.
• the remaining intemucleotide linkages are phosphodiester linkages. In embodiments, the remaining intemucleotide linkages are methylphosphonate linkages. In embodiments, 100% of the intersugar linkages of the phosphorothioate polymer backbone are phosphorothioate linkages.
• Nucleic acids can include nonspecific sequences.
• nonspecific sequence refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence.
• a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.
• An "inhibitory nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymer of nucleotide analogs) that is capable of binding to a target nucleic acid (e.g.
• a "labeled nucleic acid or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals,
• the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable label, as disclosed herein and generally known in the art.
• the level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.
• the level of expression of non-coding nucleic acid molecules may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et al, 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.
• identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
• Antibody refers to a polypeptide comprising a framework region from an
• immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
• antibodies or fragments of antibodies may be derived from different organisms, including humans, mice, rats, hamsters, camels, etc.
• Antibodies of the invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g. glycosylation, expression, antigen recognition, effector functions, antigen binding, specificity, etc.).
• a single-chain variable fragment is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids.
• the linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility.
• the linker can either connect the N- terminus of the VH with the C -terminus of the VL, or vice versa.
• the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
• Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby,
• Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison et al, PNAS USA, 81 :6851-6855 (1984), Jones et al, Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol, 44:65-92 (1988), Verhoeyen et al., Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun, 28:489-498 (1991); Padlan, Molec. Immun, 31(3): 169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No.
• an antibody-drug conjugate or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to to an antibody.
• a “therapeutic agent” as referred to herein, is a composition useful in treating or preventing a disease such as cancer.
• contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a biotin domain as described herein and a biotin-binding domain.
• contacting includes, for example, allowing a biotin domain as described herein to interact with a biotin-binding domain.
• a patient is human.
• a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor.
• the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
• the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells.
• the secondary tumor in the breast is referred to a metastatic lung cancer.
• metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
• non-metastatic cancer or subj ects with cancer that is not metastatic refers to diseases in which subj ects have a primary tumor but not one or more secondary tumors.
• Poliovirus Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Simian
• an "intracellular target” is a target, e.g., nucleic acid, polypeptide or other molecule (e.g., carbohydrate) that is located inside of a cell and is a target to which the non-cell penetrating proteins provided herein bind. Binding can be direct or indirect.
• the non-cell penetrating protein selectively binds the intracellular target.
• selectively binds, selectively binding, or specifically binding refers to the agent (e.g., a non-cell penetrating protein) binding one agent (e.g., intracellular target) to the partial or complete exclusion of other agents.
• binding is meant a detectable binding at least about 1.5 times the background of the assay method. For selective or specific binding such a detectable binding can be detected for a given agent but not a control agent. Alternatively, or additionally, the detection of binding can be determined by assaying the presence of down-stream molecules or events.
• conjugate refers to the association between atoms or molecules.
• the association can be direct or indirect.
• a conjugate between a nucleic acid and a protein can be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
• electrostatic interactions e.g. ionic bond, hydrogen bond, halogen bond
• van der Waals interactions e.g. dipole-dipole, dipole-induced dipole, London dispersion
• ring stacking pi effects
• conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
• nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
• electrophilic substitutions e.g., enamine reactions
• additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
• haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
• a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion
• the first member of the biotin binding pair and the second member of the biotin binding pair may be covalently or non-covalently attached to the non-cell penetrating protein, the phosphorothioate nucleic acid or phosphorothioate polymer backbone.
• the non- cell penetrating protein is covalently attached to the first member of the biotin binding pair (e.g., avidin, streptavidin) and the phosphorothioate nucleic acid or the phosphorothioate polymer backbone is covalently attached to the second member of the biotin binding pair (e.g., biotin).
• the cell-penetrating conjugate includes (i) a non-cell penetrating protein, (ii) a phosphorothioate nucleic acid and (iii) a non- covalent linker attaching the phosphorothioate nucleic acid to the non-cell penetrating protein.
• the non-covalent linker includes a biotin-binding domain non-covalently attached to a biotin domain and the phosphorothioate nucleic acid enhances intracellular delivery of the non-cell penetrating protein.
• polymer backbones enhances intracellular delivery of the non-cell penetrating protein.
• polymer backbones contain the same structure (i.e., contains a chain of two or more sugar residues linked together) as a nucleic acid sequence with the exception that the polymer backbone lacks the bases normally present in a nucleic acid sequence.
• cells comprising the cell penetrating conjugates.
• the non-cell penetrating protein includes a covalent reactive moiety (as deescribed abvove) and the reactive moiety is reactive with the biotin-binding domain or the biotin domain.
• the biotin-binding domain or the biotin domain includes a covalent reactive moiety and the reactive moiety is reactive with the non-cell penetrating protein.
• the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a covalent reactive moiety and the reactive moiety is reactive with the biotin- binding domain or the biotin domain.
• a cell including the cell penetrating conjugate provided herein including embodiments thereof is provided.
• the cells may include a plurality of cell penetrating conjugates.
• the cell is a cancer cell.
• the cell is a non-cancer cell.
• the cell is a T cell.
• the cell is a regulatory T cell.
• the conjugate is bound within the cell to an intracellular target.
• the second non-cell penetrating protein binds a different epitope on the intracellular target relative to the non-cell penetrating protein provided herein including embodiments thereof. In embodiments, the second non-cell penetrating protein binds a second intracellular target. In embodiments, the second non-cell penetrating protein is an antibody.
• compositions can include a single agent or more than one agent.
• the compositions further include a second non-cell penetrating protein attached to one or more phosphorothioate nucleic acids or polymer backbones through a non-covalent linker including a biotin-binding domain non-covalently bound to a biotin domain.
• the second non-cell penetrating protein binds an intracellular target. In embodiments, the second non-cell penetrating protein binds a different epitope on the intracellular target relative to the first non- cell penetrating protein. In embodiments, the second non-cell penetrating protein binds a second intracellular target. In embodiments, the first and/or second non-cell penetrating protein is an antibody. The first and second non-cell penetrating proteins can be the same protein or a different protein. [0121]
• the compositions for administration will commonly include an agent as described herein dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
• aqueous carriers e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain
• Solutions of the active compounds as free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
• compositions can be delivered via intranasal or inhalable solutions or sprays, aerosols or inhalants.
• Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays.
• Nasal solutions can be prepared so that they are similar in many respects to nasal secretions.
• the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.
• antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers, if required, may be included in the formulation.
• Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines.
• aqueous solutions for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• Aqueous solutions in particular, sterile aqueous media, are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
• Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium. Vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients, can be used to prepare sterile powders for reconstitution of sterile injectable solutions.
• the preparation of more, or highly, concentrated solutions for direct injection is also contemplated.
• DMSO can be used as solvent for extremely rapid penetration, delivering high concentrations of the active agents to a small area.
• Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
• Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
• auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
• auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
• auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents
• a control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to disease, or prior to treatment.
• diagnosis refers to a relative probability that a disease (e.g. an autoimmune, inflammatory autoimmune, cancer, infectious, immune, or other disease) is present in the subject.
• comparing correlating and associated, in reference to determination of a disease risk factor, refers to comparing the presence or amount of the risk factor (e.g., amount of intracellular target of a disease) in an individual to its presence or amount in persons known to suffer from, or known to be at risk of disease, or in persons known to be free of disease, and assigning an increased or decreased probability of having/devel oping the disease to an individual based on the assay result(s).
• the risk factor e.g., amount of intracellular target of a disease
• binding is detereming by an imaging method or system.
• the cell penetrating conjugates provided herein including embodiments thereof can also be used in imaging applications or other applications for analyzying intracellular target levels and/or activities.
• the provided cell penetrating conjugates can be used for in vitro or in vivo imaging of intracellular targets of interest.
• the cell penetrating conjugates are used for live cell imaging.
• live cell imaging can be used to monitor intracellular target distribution and/or dynamics inside living cells and is also applicable to monitoring target interactions.
• the method includes administering to the subject a second non-cell penetrating protein including a second non-covalent linker attaching one or more
• the disease is a metabolic disorder. In embodiments, the disease is a metabolic disorder.
• compositions of the present invention can also be delivered as microspheres for slow release in the body.
• microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).
• the second non-cell penetrating protein binds a different epitope on the intracellular target relative to the non-cell penetrating protein provided herein including embodiments thereof. In embodiments, the second non-cell penetrating protein binds a second intracellular target. In embodiments, the second non-cell penetrating protein is formulated as a pharmaceutical composition including a second non-cell penetrating protein and a pharmaceutically acceptable carrier. In embodiments, the second non-cell penetrating protein is an antibody. In
• the U251 cells were treated with 10 ⁇ g/ml of the indicated modified anti-STAT3 antibodies and were incubated for one hour at 37°C. After the incubation and two washing steps with PBS (2ml) the cells were lysed via incubation (30min.) on ice in 500 ⁇ 1 RIPA buffer, followed by vigorous vortexing. The full cell lysates were harvested by collecting the supernatant after a
• the membrane was blocked with 10% BSA/TBS-T (0.1 % Tween20) for one hour at room temperature on a shaking platform. Afterwards the membrane was incubated with the primary antibody (anti-STAT3 1 : 1.000 in TBS-T) overnight at 4°C on a shaking platform. After three washing steps (15min., lOmin., lOmin. with TBS-T) with TBS-T the membrane were incubated with ECLTM Anti-Rabbit IgG, horseradish peroxidase (1 : 10.000 in TBS-T) at 4°C on a shaking platform for 2-4 hours. The detection took place using the
• FIG. 7A shows flow cytometry analysis to detect delivered antibodies, using an anti-rabbit antibody.
• U251 cells human glioma stem cells
• modified anti-STAT3 antibodies rabbit conjugated to indicated PS DNA -oligos: (80mer) (pointed line), PS-oligo DNA (40mer) (dashed line) and PS-oligo DNA (20mer) (red area) via Streptavidin/Biotin.
• FIG. 7A lower panel shows same as FIG.
• FIG. 9A depicts alternative Western blotting analysis of U251 cells treated with modified anti-STAT3 antibodies conjugated to PS-oligo DNA (20mer) (first lane), PS-oligo RNA (20mer) (second lane), PO-polymer (20mer) (third lane) and PS-polymer (20mer) (fourth lane) via Strepavidin/Biotin.
• the U251 cells were treated with lC ⁇ g/ml of the particular modified anti-STAT3 antibody and were incubated for one hour at 37°C.
• Embodiment 7 The cell-penetrating conjugate of any one of embodiments 1-6, wherein a plurality of biotin-binding domains are attached to said non-cell penetrating protein.
• Embodiment 9 The cell-penetrating conjugate of embodiment 8, wherein said biotin domain is covalently attached to said phosphorothioate nucleic acid.
• Embodiment 10 The cell-penetrating conjugate of embodiment 8 or 9, wherein a plurality of phosphorothioate nucleic acids are attached to said biotin domain.
• Embodiment 13 The cell-penetrating conjugate of embodiment 11, wherein said phosphorothioate nucleic acid is about 20 nucleic acid residues in length.
• Embodiment 20 The cell penetrating conjugate of any one of embodiments 16 to 19, wherein the antibody is a humanized antibody.
• Embodiment 21 The cell penetrating conjugate of any one of embodiments 1 to 20, wherein said non-cell penetrating protein binds an intracellular target.
• Embodiment 29 The cell penetrating conjugate of any one of embodiments 1 to 28, wherein said cell penetrating conjugate is bound to an intracellular target.
• Embodiment 33 The method of embodiment 30, wherein said first member of said biotin binding pair is a biotin domain.
• Embodiment 40 The pharmaceutical composition of embodiment 38, wherein said second non-cell penetrating protein binds an intracellular target.
• Embodiment 44 A kit comprising the cell penetrating conjugate of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 37 and instructions for use.
• Embodiment 45 The kit of embodiment 44, further comprising a second non-cell penetrating protein comprising a second non-covalent linker attaching one or more
• Embodiment 46 The kit of embodiment 45, wherein said conjugate of any one of embodiments 1 to 29 and said second non-cell penetrating protein are in separate containers.
• Embodiment 49 The kit of any one of embodiments 45 to 48, wherein said second non- cell penetrating protein is formulated as a pharmaceutical composition comprising the second non-cell penetrating protein and a pharmaceutically acceptable carrier.
• Embodiment 50 The kit of any one of embodiments 45 to 49, wherein the second non- cell penetrating protein is an antibody.
• Embodiment 51 A method of delivering a non-cell penetrating protein into a cell comprising contacting the cell with said cell penetrating conjugate of any one of embodiments 1 to 29.
• Embodiment 54 A method of treating a disease in a subject in need thereof, said method comprising administering to a subject an effective amount of the cell penetrating conjugate of any one of embodiments 1 to 29, thereby threating the disease in said subject.
• Embodiment 60 The method of any one of embodiments 55 to 58, wherein said conjugate of any one of embodiments 1 to 29 and said second non-cell penetrating protein are administered sequentially.
• Embodiment 61 The method of any one of embodiments 55 to 60, wherein said second non-cell penetrating protein is an antibody.

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