Patent Grant
12077795
The present invention relates to a protein catalyst for the attachment of diverse chemical matter, specifically oligonucleotides, to a specific amino acid in a polypeptide substrate.
All references cited herein are expressly incorporated herein by reference in their entirety.
Site-specific selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally occurring post-translational modifications, for creating antibody-drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics, and protein-protein interactions, and for the preparation of protein-polymer conjugates. One approach for protein labeling is to incorporate biorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated sites then support chemoselective reactions, since reactions may be defined that are inert to normal biological materials, and occur selectively when the biorthogonal component is present. Known enzymes for site-specific ligation include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase.
Proteins, comprised of varying numbers of 20 distinct amino acid residues, arranged in a specific sequence, are the primary mediators of biological processes in all organisms, from single cell bacteria to humans. Techniques to manipulate the function of proteins can therefore find important applications in fundamental science as well as medicine and engineering. For example, the capacity to attach therapeutic chemical matter to specific amino acid residues in an antibody can pave the way for targeted therapeutics (i.e. antibody-drug conjugates). In addition, techniques to attach fluorophores or other optical probes to specific amino acid residues of an enzyme can prove useful for investigating the protein's spatial and temporal function in a specific biological process.
Joining together chemical matter with a protein (i.e. conjugation) requires at a minimum two reactive functional groups, such as a nucleophile and an electrophile, that, when combined in solution, chemically unite. Because proteins are metastable, suitable conjugation chemistry must involve functional groups that react together selectively without appreciable side-reactions; tolerate the presence of water, salts, and buffers; proceed at reasonable rates at ambient temperature; and progress to near completion so as to minimize post-reaction workup of the conjugated protein.
There are several proteins with conjugation activity that have been developed commercially. Prominent examples: Halotag (Promega) www.promega.com/products/pm/halotag-technology/halotag-technology/SNaP tag (New England Biolabs) www.neb.com/applications/protein-analysis-and-tools/proteinlabeling/protein-labeling-snap-clip Biotin ligase (Avidity) www.avidity.com/technologies/vitrobiotinylation-avitag-enzyme Sfp phosphopantetheinyltransferase (New England Biolabs) www.neb.com/products/p9302-sfp-synthase.
Of specific interest here is the conjugation of proteins to nucleic acids. Protein-DNA conjugates have been sought for fundamental and applied studies. In his 2010 review Niemeyer, Christof M. “Semisynthetic DNA-protein conjugates for biosensing and nanofabrication.” Angewandte Chemie International Edition 49, no. 7 (2010): 1200-1216 (www.ncbi.nlm.nih.gov/pubmed/20091721), Niemeyer identified a number of emerging areas, including bioanalytics (i.e immunoPCR); DNA directed immobilization of proteins (biochips); nanofabrication of protein assemblies (DNA arrays); and synthesis of medicinal nanoparticles bearing therapeutic proteins and peptides.
Current methods to conjugate proteins with nucleic acids depend on “spontaneous” chemical conjugation chemistry, such as disulfide bond formation, as opposed to conjugation catalyzed by a biomolecule. No enzyme has been fully described that can directly conjugate proteins with nucleic acids. Rather, methods in current use require installing reactive functional groups on the protein and separately, on the nucleic acid (Neimeyer, www.ncbi.nlm.nih.gov/pubmed/20091721). The chemically modified protein and nucleic acid are combined in a single tube and allowed to react. Often, these bimolecular reactions proceed slowly, requiring 24 h or more, and generate modest yields, see for example (Barbuto, Scott, Juliana Idoyaga, Miguel Vila-Perelló, Maria P. Longhi, Gaelle Breton, Ralph M. Steinman, and Tom W. Muir. “Induction of innate and adaptive immunity by delivery of poly dA: dT to dendritic cells.” Nature chemical biology 9, no. 4 (2013): 250-256, www.ncbi.nlm.nih.gov/pubmed/23416331). More recently, strategies have emerged that make use of activated esters as well as click chemistry, and appear to increase the speed conjugation; however they suffer from a lack of specificity with respect to the site (i.e. amino acid residue) of protein-nucleic acid conjugation (www.ncbi.nlm.nih.gov/pubmed/26947912).
See (each of which is expressly incorporated herein by reference in its entirety):
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of compounds.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. As used herein the following terms have the following meanings.
As used herein, the term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by (+) or (−) 10%, 5% or 1%.
As used herein, the term “administration” may be effected in one dose, continuously or intermittently or by several subdoses which in the aggregate provide for a single dose. Dosing can be conducted throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue. Non-limiting examples of route of administration include intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, nasal spray and other mucosal delivery (e.g. transmucosal delivery), intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, intradermal injection, electroincorporation (e.g., with electroporation), ultrasound, jet injector, oral and topical patches.
A “therapeutic agent,” as used herein, may be a molecule, or compound that is useful in treatment of a disease or condition. A “therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose” is the amount of a compound that produces a desired therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, producing a desired physiological effect, or allowing imaging or diagnosis of a condition that leads to treatment of the disease or condition. The precise therapeutically effective amount is the amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.
As used herein, “in combination” or “in combination with,” when used herein in the context of multiple agents, therapeutics, or treatments, means in the course of treating the same disease or condition in a subject administering two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof. This includes simultaneous administration (or “coadministration”), administration of a first agent prior to or after administration of a second agent, as well as in a temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, treatment regimens or treatment modalities. Further, the administration of the two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration.
“Treating” or “treatment” of a condition, disease or disorder may refer to preventing the condition, disease or disorder, slowing the onset or rate of development of the condition, disease or disorder, reducing the risk of developing the condition, disease or disorder, preventing or delaying the development of symptoms associated with the condition, disease or disorder, reducing or ending symptoms associated with the condition, disease or disorder, generating a complete or partial regression of the condition, disease or disorder, or some combination thereof. Examples of neoplastic diseases or disorders include colorectal cancer, osteosarcoma, non-small cell lung cancer, breast cancer, ovarian cancer, glial cancer, solid tumors, metastatic tumor, acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancers, ductal carcinoma in situ, endometrial cancer, esophageal cancer, eye cancer, intraocular, retinoblastoma, metastatic melanoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hepatoma, Hodgkin lymphoma, hypopharyngeal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, melanoma, squamous neck cancer, mouth cancer, multiple myeloma, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic carcinoma, papillary carcinomas, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors, pineoblastoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, salivary gland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma, squamous cell carcinoma, Sezary syndrome, skin cancer, Merkel cell carcinoma, testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroid cancer, urethral cancer, endometrial cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.
“Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. Tumor or cancer status may also be assessed by sampling for the number, concentration or density of tumor or cancer cells, alone or with respect to a reference. In accordance with the practice of the invention, inhibiting a tumor may be measured in any way as is known and accepted in the art, including complete regression of the tumor(s) (complete response); reduction in size or volume of the tumor(s) or even a slowing in a previously observed growth of a tumor(s), e.g., at least about a 10-30% decrease in the sum of the longest diameter (LD) of a tumor, taking as reference the baseline sum LD (partial response); mixed response (regression or stabilization of some tumors but not others); or no apparent growth or progression of tumor(s) or neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum LD since the treatment started (stable disease).
“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. “oligonucleotide” refers to a nucleotide having a chain length of less than 250 nucleotides. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides, and/or their analogs, including epigenetically modified variants, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction.
A “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), pets (such as cats, dogs and horses), primates, mice and rats.
An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
A “therapeutically effective amount” of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
According to the present invention, where administration includes a pharmaceutical formulation, preferably the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient (also referred to herein as a therapeutic agent).
The compositions of the invention can be administered by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a nontoxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.
In human therapy, compositions of the invention may be administered alone but may generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
In embodiments of the present invention in which polypeptides or polynucleotides of the invention are administered parenterally, such administration can be, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intracisternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Therapeutic formulations may be prepared for storage by mixing an active component having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
The formulation may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington: The Science and Practice of Pharmacy 20th edition (2000).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. When appropriate, chemical or radiation sterilization method may be used.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated immunoglobulins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. The formulation may also be an immediate-release formulation. The formation may also be a combination of an immediate-release formulation and a sustained-release formulation.
The medicaments and/or pharmaceutical compositions may be present in a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
The agents, medicaments and pharmaceutical compositions of the invention may be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.
In human therapy, the agents, medicaments and pharmaceutical compositions of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, the agents, medicaments and pharmaceutical compositions of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed- or controlled-release applications. Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agents, medicaments and pharmaceutical compositions of the invention may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The agents, medicaments and pharmaceutical compositions can be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The medicaments and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
For oral and parenteral administration to human patients, the daily dosage level of the agents, medicaments and pharmaceutical compositions of the invention will usually be from 10 μg to 500 mg per adult per day administered in single or divided doses.
The agents, medicaments and pharmaceutical compositions of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of an agent of the invention and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each metered dose or “puff” contains at least 1 mg of an agent of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
Alternatively, the agents, medicaments and pharmaceutical compositions can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, gel, ointment or dusting powder. The agents, medicaments and pharmaceutical compositions of the invention may also be transdermally administered, for example, by the use of a skin patch, ointment, cream or lotion. For application topically to the skin, the agents, medicaments and pharmaceutical compositions of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
They may also be administered by the ocular route, particularly for treating diseases of the eye. For ophthalmic use, the agents, medicaments and pharmaceutical compositions can be formulated as soluble suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Agents may be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used, for example as described in the accompanying Examples. For in vitro applications, formulations may comprise a lower concentration of a compound of the invention.
Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents.
The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present agents that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
Autoproteolytic cleavage is a critical step in the maturation of Hedgehog (Hh) proteins, where a precursor form of Hh is activated for nucleophilic attack by cholesterol. No cofactors, energy source or accessory proteins are required for this unusual cholesterolysis event; instead, all catalytic activity resides in a ˜26 kDa segment of the Hh precursor.
This autocatalytic element, termed hedgehog steroyl transferse I (HST-I) or hedgehog terminal transferase (HTTase), or HhC, may be used as a tool for protein labeling. HST-I is modular; and its activity is retained when fused to the C-terminus of heterologous peptides and proteins.
Using an optical assay developed to monitor HST-I, heterologous proteins can be covalently modified at their C-terminus by cholesterol with a t1/2 of 10-20 min at pH 7.1, 25 degrees Celsius. During the reaction, HST-I is liberated from the heterologous protein, rendering the reaction “traceless”, i.e., the product does not contain required traces of the HST-I component.
Sterols other than cholesterol may be used as substrates. In addition to cholesterol, HST-I will also ligate proteins to sterol derivatives equipped with fluorescent reporters, alkynes, and biotin, among other functional groups.
Typically, cholesterol modified proteins partition to eukaryotic cell membranes.
See,
Hedgehog terminal transferase (HST-I) thus provides a tool which can be used to covalently conjugate virtually any protein with a variety of small molecules, from fluorophores to therapeutics. HST-I is a water soluble protein that catalyzes protein conjugation in <60 min in the absence accessory proteins.
Features of protein-conjugation with HST-I include: (1) two-component labeling kinetics (2) site-specific, stoichiometric modification (3) broad substrate tolerance and (4) labeling that is nearly traceless. The last feature is particularly noteworthy as existing protein conjugation methods require addition to the target protein of anywhere from 4 to 296 amino acids. That residual sequence can compromise function or engender an immune response during therapeutic application. Conjugation with HST-I requires addition to the target protein of a single glycine residue only (unless it is already present and available).
A general approach is provided for biocatalytic protein-small molecule conjugation, where the reaction is site-specific (C-terminus), stoichiometric (1:1), and nearly traceless (1 or 2 amino acid “scar”). The catalyst for this reaction, called HST-I, has the amino acid sequence (from Drosophila melanogaster), as follows:
G{circumflex over ( )}CFTPESTALLESGVRKPLGELSIGDRVLSMTANGQAVYSEVILFMDRN
The Hedgehog sterol transferase (HST-I) from Drosophila melanogaster HST-I conjugates the first amino acid (glycine, GA) to a sterol through a carboxylic acid ester. Other steroyl transferases and/or hedgehog terminal transferases with homology to HST-I may also be used, in similar fashion (For related sequences see: Burglin, Thomas R. “The Hedgehog protein family.” Genome biology 9, no. 11 (2008): 241, www.ncbi.nlm.nih.gov/pubmed/19040769). Homologous proteins are endogenous to multicellular organisms, vertebrates and invertebrates.
The native function of HST-I is to covalently couple the carboxyl terminus of a specific protein (substrate A) to cholesterol (substrate B). HST-I exhibits broad substrate tolerance toward substrate A and toward substrate B. Thus, proteins with no homology to the native protein substrate (substrate A) can be modified at their carboxyl terminus by HST-I; in addition, sterols other than cholesterol (substrate B), derivatized with a variety of functional groups can serve as the substrate for HST-I.
In each of these cases, the core steroid nucleus serves as the generic “linker” recognized by HST-I that ultimately joins protein to functional group.
A genetically engineered HST-I or HST-I derivative may be used, whose conjugation activity can be regulated by an exogenous trigger (redox, light); exhibits different solution properties (e.g., higher thermal stability; greater aggregation resistance); as well as HST-I derivatives that have been engineered to accept synthetic sterols with alterations to the fused ring system of sterols. The HST-I may also be switched, such as by pH, ligand or ionic binding, or the like.
HST-I has utility, particularly in the application areas of protein therapeutics (e.g. antibody-drug conjugates/protein-nucleic acid conjugates), protein detection, and protein immobilization. As one example, HST-I could be used to attach a therapeutic compound, or an imaging agent, to the caboxyl terminus of an antibody so as to target that cargo to a cancer cell bearing the appropriate antigen/receptor. In another embodiment, HST-I could be used to attach polypeptides to nanoparticles for biological or biophysical studies. HST-I can also be used to create protein-nucleic acids conjugates to enhance cellular delivery of therapeutic RNA or natural or synthetic polynucleotides.
Some examples include antibody-drug conjugates; protein-fluorophore labeling for imaging; protein immobilization for diagnostic applications (sensors); membrane-targeted therapeutics (hormones/antibacterials/inhibitors of membrane-bound enzymes); and membrane-targeted protein probes (GFP, etc.).
There are similarities and key distinctions of HST-I compared with other biocatalysts for protein conjugation. Conjugation by HST-I is residue specific; it is active in physiological buffer at room temperature; the kinetics are relatively fast (half time ˜1 h or less); and a variety of protein substrates (substrate A) can be labeled with a broad range of small molecules (substrate B).
The application of HST-I generally correspond to the applications of Sortase enzyme and farnesyl transferase. See, U.S. Pat. Nos. 6,544,772; 6,620,585; 6,773,706; 6,790,448; 6,838,239; 6,841,154; 6,896,887; 6,908,994; 6,936,252; 6,994,982; 7,067,248; 7,067,621; 7,067,639; 7,101,692; 7,125,698; 7,179,458; 7,183,101; 7,195,763; 7,238,489; 7,270,969; 7,312,076; 7,348,420; 7,371,719; 7,384,775; 7,390,526; 7,452,679; 7,455,992; 7,456,011; 7,485,710; 7,491,690; 7,534,761; 7,534,876; 7,538,209; 7,541,039; 7,544,661; 7,554,021; 7,615,616; 7,632,515; 7,635,487; 7,648,708; 7,683,025; 7,709,009; 7,713,534; 7,722,888; 7,731,978; 7,745,708; 7,754,467; 7,763,420; 7,771,728; 7,771,731; 7,776,553; 7,776,589; 7,785,608; 7,803,765; 7,820,184; 7,829,681; 7,833,791; 7,838,010; 7,838,491; 7,850,974; 7,851,445; 7,858,357; 7,888,075; 7,897,367; 7,927,864; 7,935,804; 7,939,087; 7,955,604; 7,960,505; 7,960,533; 7,968,100; 7,968,297; 7,968,683; 8,007,803; 8,007,811; 8,025,885; 8,025,890; 8,038,990; 8,039,005; 8,063,014; 8,076,295; 8,088,611; 8,101,194; 8,105,612; 8,110,199; 8,124,107; 8,124,583; 8,128,936; 8,137,673; 8,138,140; 8,148,321; 8,241,642; 8,252,546; 8,263,642; 8,280,643; 8,287,885; 8,318,908; 8,323,660; 8,329,195; 8,372,411; 8,377,446; 8,399,651; 8,409,589; 8,431,139; 8,445,426; 8,450,271; 8,475,809; 8,524,241; 8,529,913; 8,557,961; 8,563,001; 8,563,006; 8,563,007; 8,568,735; 8,574,597; 8,575,070; 8,580,923; 8,580,939; 8,591,899; 8,592,375; 8,598,342; 8,609,106; 8,617,556; 8,632,783; 8,647,835; 8,652,800; 8,663,631; 8,663,926; 8,669,226; 8,673,860; 8,679,505; 8,680,050; 8,703,717; 8,709,431; 8,709,436; 8,709,760; 8,710,188; 8,715,688; 8,716,448; 8,722,354; 8,748,122; 8,754,198; 8,758,765; 8,772,049; 8,778,358; 8,795,965; 8,795,983; 8,808,699; 8,821,894; 8,822,409; 8,835,187; 8,835,188; 8,840,906; 8,841,249; 8,853,382; 8,858,957; 8,859,492; 8,865,479; 8,871,204; 8,871,445; 8,883,788; 8,889,145; 8,889,150; 8,889,356; 8,927,230; 8,932,814; 8,933,193; 8,933,197; 8,937,167; 8,940,501; 8,945,542; 8,945,588; 8,945,589; 8,945,855; 8,946,381; 8,957,021; 8,961,979; 8,975,232; 8,980,284; 8,980,824; 8,986,710; 8,993,295; 9,005,579; 9,050,374; 9,056,912; 9,062,299; 9,068,985; 9,079,946; 9,079,952; 9,080,159; 9,090,677; 9,095,540; 9,102,741; 9,107,873; 9,109,008; 9,114,105; 9,127,050; 9,128,058; 9,129,785; 9,132,179; 9,132,182; 9,134,304; 9,150,626; 9,156,850; 9,168,293; 9,168,312; 9,181,297; 9,181,329; 9,182,390; 9,205,142; 9,212,219; 9,217,157; 9,221,882; 9,221,886; 9,221,902; 9,234,012; 9,238,010; 9,243,038; 9,249,211; 9,266,925; 9,266,943; 9,266,944; 9,267,127; 9,315,554; 9,340,582; 9,340,584; 9,353,160; 9,371,369; 9,376,672; 9,382,289; 9,388,225; 9,393,294; 9,394,092; 9,399,673; 9,403,904; 9,404,922; 9,405,069; 9,408,890; 9,409,952; 9,416,171; 9,434,774; 9,441,016; 9,458,228; 9,458,229; 9,463,431; 20020028457; 20030021789; 20030022178; 20030087864; 20030091577; 20030099940; 20030153020; 20030180816; 20030185833; 20030186851; 20030228297; 20040091856; 20040101919; 20040110181; 20040126870; 20040167068; 20040230033; 20040236072; 20050002925; 20050003510; 20050037444; 20050048545; 20050069984; 20050106648; 20050112612; 20050175581; 20050203280; 20050207995; 20050220788; 20050233396; 20050276814; 20060073530; 20060074016; 20060078901; 20060115491; 20060135416; 20060165716; 20060177462; 20060188975; 20060194226; 20060198852; 20060234222; 20060246080; 20060257413; 20060263846; 20060269538; 20060275315; 20070003667; 20070026011; 20070031832; 20070053924; 20070059295; 20070082006; 20070082007; 20070082866; 20070117197; 20070128210; 20070128211; 20070128229; 20070172495; 20070190029; 20070190063; 20070207170; 20070207171; 20070218075; 20070248581; 20070253964; 20070258955; 20070286866; 20080031877; 20080038287; 20080050361; 20080064079; 20080089899; 20080171059; 20080175857; 20080220441; 20080248522; 20080254070; 20080260768; 20090022753; 20090035780; 20090041744; 20090075839; 20090088337; 20090088372; 20090092582; 20090117113; 20090130115; 20090148408; 20090155304; 20090176967; 20090202578; 20090202593; 20090214476; 20090214537; 20090214584; 20090239264; 20090297548; 20090297549; 20090305252; 20090317420; 20090317421; 20100004324; 20100009917; 20100055761; 20100064393; 20100068220; 20100074923; 20100098789; 20100105865; 20100119534; 20100150943; 20100152054; 20100183614; 20100184624; 20100196524; 20100221288; 20100227341; 20100239554; 20100247561; 20100255026; 20100256070; 20100260706; 20100260790; 20100267053; 20100278740; 20100279328; 20100297183; 20100303864; 20100323956; 20110020323; 20110020385; 20110020402; 20110020900; 20110046008; 20110046060; 20110046061; 20110076299; 20110077199; 20110091956; 20110097360; 20110104168; 20110110982; 20110124520; 20110129935; 20110150918; 20110151053; 20110172146; 20110177976; 20110183863; 20110189187; 20110189236; 20110189664; 20110206616; 20110206676; 20110206692; 20110243976; 20110243977; 20110245480; 20110262477; 20110263501; 20110275132; 20110281745; 20110281764; 20110288005; 20110311536; 20110312881; 20110318339; 20110321183; 20120015379; 20120034261; 20120058906; 20120064103; 20120064104; 20120076814; 20120083599; 20120093840; 20120093850; 20120100174; 20120100569; 20120107340; 20120114686; 20120121643; 20120122123; 20120142682; 20120149590; 20120149710; 20120157665; 20120171211; 20120172303; 20120178691; 20120189649; 20120201844; 20120207778; 20120237536; 20120244189; 20120251568; 20120263701; 20120263703; 20120270797; 20120282247; 20120282289; 20120282670; 20120282700; 20120289454; 20120294880; 20120301428; 20120301433; 20120301496; 20120308595; 20120309679; 20120309701; 20120316071; 20130011386; 20130011428; 20130017997; 20130034575; 20130034847; 20130039884; 20130045211; 20130064845; 20130071416; 20130072420; 20130084648; 20130089525; 20130101665; 20130122043; 20130136746; 20130136761; 20130143955; 20130157281; 20130165389; 20130171183; 20130177940; 20130184177; 20130189287; 20130209494; 20130216568; 20130217592; 20130217612; 20130230550; 20130236419; 20130243818; 20130253175; 20130259889; 20130260404; 20130261293; 20130288266; 20130288267; 20130289251; 20130289253; 20130296257; 20130316946; 20130323819; 20130330335; 20130338030; 20130338047; 20130344010; 20140004138; 20140011709; 20140017764; 20140030697; 20140037650; 20140037669; 20140051834; 20140057317; 20140065171; 20140073639; 20140105818; 20140113832; 20140128289; 20140147873; 20140154286; 20140154287; 20140155319; 20140161915; 20140162949; 20140170702; 20140178425; 20140179006; 20140186265; 20140186327; 20140186350; 20140186354; 20140186358; 20140189896; 20140193438; 20140206840; 20140213515; 20140227295; 20140227298; 20140234972; 20140235828; 20140243280; 20140248702; 20140249296; 20140255470; 20140273231; 20140287509; 20140301974; 20140302084; 20140308318; 20140310830; 20140315314; 20140328819; 20140329706; 20140329750; 20140335095; 20140348868; 20140371136; 20140377289; 20150004155; 20150005233; 20150005481; 20150010566; 20150023879; 20150023959; 20150030594; 20150031134; 20150031563; 20150031604; 20150037359; 20150037421; 20150037828; 20150038421; 20150045535; 20150050717; 20150051082; 20150056239; 20150056240; 20150071957; 20150079132; 20150079681; 20150086576; 20150087545; 20150093406; 20150093413; 20150104468; 20150118183; 20150118264; 20150132324; 20150132335; 20150132339; 20150139984; 20150152134; 20150158929; 20150165062; 20150166640; 20150168405; 20150174130; 20150182588; 20150184142; 20150185216; 20150197538; 20150197734; 20150203834; 20150210756; 20150216960; 20150231228; 20150232518; 20150232541; 20150232560; 20150232561; 20150241440; 20150246024; 20150253335; 20150258210; 20150259389; 20150259397; 20150259431; 20150266943; 20150273040; 20150273042; 20150274800; 20150284452; 20150284477; 20150290362; 20150291704; 20150305361; 20150306212; 20150306218; 20150309021; 20150315248; 20150320882; 20150329590; 20150335724; 20150338579; 20150343051; 20150344862; 20150346195; 20150368322; 20150374811; 20150376266; 20160000895; 20160002338; 20160002346; 20160002645; 20160018397; 20160022776; 20160022833; 20160025740; 20160032346; 20160038581; 20160040158; 20160041157; 20160045885; 20160052982; 20160068583; 20160068589; 20160068591; 20160069894; 20160073671; 20160074497; 20160074498; 20160082046; 20160090351; 20160090404; 20160097773; 20160102137; 20160102332; 20160102344; 20160108091; 20160114046; 20160115222; 20160115488; 20160115489; 20160122405; 20160122451; 20160122707; 20160123991; 20160129101; 20160130299; 20160136298; 20160137698; 20160137711; 20160137720; 20160146786; 20160146794; 20160158335; 20160166634; 20160168232; 20160175412; 20160175441; 20160178627; 20160184421; 20160185791; 20160185817; 20160185828; 20160193355; 20160194363; 20160194410; 20160194627; 20160199454; 20160199510; 20160200742; 20160206566; 20160208233; 20160213744; 20160220686; 20160222096; 20160230216; 20160244747; 20160244784; 20160251409; 20160257749; 20160257932; 20160264624; 20160271268; 20160279192; 20160279257; 20160280748; 20160282369; 20160287734; 20160297854; each of which is expressly incorporated herein by reference. Likewise, applications provided in U.S. 2015/0329568, expressly incorporated herein by reference in its entirety, may be employed as appropriate.
There are two key advantages of HST-I over sortase and farnestyl transferase biocatalysts for protein conjugation: First, HST-I allows a one-pot, bi-molecular conjugation, whereas existing sortase and farnesyl transferase bioconjugations require 3 component reactions. Second, the conjugation reaction with HST-I is nearly traceless, as the HST-I cleaves itself from the target protein during conjugation. The bi-molecular labelling (i) stems from the fact that the catalyst, HST-I, is fused to the protein substrate (substrate A), thus only the modifier (substrate B) needs to be added to the “pot” to initiate conjugation. No cofactors or accessory proteins are required.
Existing methods that require at least three separate components (the protein of interest; the small molecule; and the conjugation catalyst). Three component coupling reactions often proceed slowly, and require excess reactant concentration to drive conjugation to completion.
The traceless feature arises from the fact that the protein of interest is liberated from HST-I upon labelling. Existing chemical labeling methods require addition to the protein of anywhere from 4 to 296 amino acid residues to allow recognition by the conjugation catalyst. That residual “scar” may compromise stability, perturb protein-protein association, or engender an immune response, which would be a serious concern for therapeutic applications, in contrast to the single glycine residue required by HST-I.
Labeling is restricted to a protein's carboxyl terminus.
The sterol moiety that functions as the generic linker is typically hydrophobic, which could compromise solubility of the labeled protein if a water-soluble/hydrophilic product is required.
The technology does require fusion of the protein of interest to HST-I, which is typically performed by genetically engineering the protein of interest fused to the HST-I, though other mechanisms of ligation may be employed.
The catalytic activity of HST-I, i.e., Hedgehog sterol transferase (HST-I, 26 kDa) does not need the entire hedgehog protein, 46 kDa, but rather only the C-terminal portion of the hedgehog protein. A signaling protein, HhN, is in the adjacent N-terminal region of the hedgehog protein, is not required for catalytic activity.
The native function of HST-I in Drosophila, and in other multicellular organisms, is to conjugate cholesterol to the last residue of HhN, which is invariably glycine. The lipid becomes joined to the carboxyl group of glycine through an ester (www.ncbi.nlm.nih.gov/pubmed/8824192). Conjugation by HST-I releases the cholesterol-modified protein, thereby generating two polypeptides; HhN-cholesterol and HST-I.
In vitro HST-I exhibits broad substrate tolerance. For example, conjugation proceeds even when HST-I is translationally fused to heterologous proteins. Examples of heterologous proteins include fluorescent proteins, enzymes, carbohydrate binding proteins, and peptides. These proteins, with no homology to the native substrate polypeptide, HhN, undergo efficient conjugation at a C-terminal glycine residue catalyzed by HST-I. Derivatives of cholesterol modified with a variety of chemical matter, including oligonucleotides, serve as suitable substrates for HST-I. In these non-native conjugation events, the steroid appears to serves as a kind of “molecular handle” recognized by HST-I and ultimately activated by HST-I so as to react with the final residue of the heterologous protein.
Advantages of conjugation of a nucleic acid with a protein of interest (POI), using HST-1 and a steroylated nucleic acid include:
Example sterols and derivatized sterols include:
Formulas I and II are metal chelators, and Formulas III and IV are fluorophores. Formula V is a steroylated nucleic acid.
Sterol derivatives may be prepared, for example, through a process as follows:
For example, derivatives may be prepared in one-pot by reductive amination of pregnenolone with propargylamine. Diastereomers are separable by silica gel chromatography.
It is therefore an object to provide a method comprising: providing a purified fusion protein comprising a C-terminal steroyl transferase activity from a hedgehog protein, an intervening electrophilic amino acid, and an N-terminal polypeptide; reacting the purified fusion protein with a substrate for the steroyl transferase activity comprising a fused sterol or stanol ring system having a nucleophilic substituent of the A ring ligated to at least one nucleic acid, to thereby cleave the fusion protein to release the C-terminal steroyl transferase activity in solution and covalently link the nucleophilic substituent of the A ring to the electrophilic amino acid.
It is also an object to provide a method for generating a polypeptide-nucleic acid conjugate, comprising: providing a polypeptide; providing a steroylated-nucleic acid; providing a protein catalyst adapted to link the polypeptide through a sterol moiety to the steroylated-nucleic acid; and reacting the steroylated-nucleic acid and the polypeptide, catalyzed by the protein catalyst, to form a covalently linked polypeptide-steroyl-nucleic acid conjugate.
The protein catalyst may comprise a hedgehog sterol transferase activity.
The polypeptide and the protein catalyst may be provided as a fusion protein.
The reacting may disassociate the protein catalyst from the polypeptide-nucleic acid conjugate. The protein catalyst may be linked to the polypeptide through a glycine, or other suitable electrophilic amino acid.
The fusion protein may comprise a C-terminal portion having the hedgehog sterol transferase activity, an intervening glycine, and an N-terminal portion comprising the polypeptide.
The steroylated-nucleic acid may comprise a canonical fused sterol or stanol ring system, a nucleophilic group at the 3-position of the A-ring of the fused sterol or stanol ring system. For example, beta or alpha stereochemistry ring systems may be employed, and an oligonucleotide attached through a linker to the sterol or stanol ring system, e.g., through a D-ring or other ring. The steroylated-nucleic acid may comprise at least one sterol molecule joined covalently to a nucleic acid polymer.
The polypeptide may have a length of between 2 amino acids and 500 amino acids. The polypeptide may have glycine as a last amino acid residue, linked to the protein catalyst having the hedgehog sterol transferase activity.
The protein catalyst may be of, or related to, a hedgehog-family of proteins. Homologous proteins have been identified by sequence alignments (Burglin, Thomas R. “The Hedgehog protein family.” Genome biology 9, no. 11 (2008): 241, www.ncbi.nlm.nih.gov/pubmed/19040769). The protein catalyst and the polypeptide may be expressed from a genetically engineered chimeric gene. The protein catalyst and the polypeptide may be expressed from the genetically engineered chimeric gene in an organism lacking sterols. The fusion chimera polypeptide may also be produced in cell-free systems, e.g., in vitro translation. The protein catalyst may be configured to react with a last residue of the polypeptide through translation fusion. The protein catalyst may be configured to chemically link a C-terminal residue, typically glycine, of the polypeptide with the steroylated-nucleic acid. The protein catalyst may have a substrate affinity for the steroylated-nucleic acid. The protein catalyst and the polypeptide may be associated prior to the reacting, and the protein catalyst and the polypeptide may be disassociated subsequent prior to the reacting to form the polypeptide-nucleic acid conjugate.
A composition is provided according to any of the foregoing.
A kit is provided, comprising: a polypeptide comprising a C-terminal steroyl transferase activity, an N-terminal peptide, and an intervening electrophilic residue, typically glycine; and a steroylated-nucleic acid, the polypeptide and the steroylated-nucleic acid being configured to react in solution to ligate the steroylated-nucleic acid to the N-terminal peptide through the glycine, and to disassociate the C-terminal steroyl transferase activity from the ligated steroylated-nucleic acid and N-terminal peptide. The C-terminal steroyl transferase activity may correspond to a hedgehog protein steroyl transferase activity.
The polypeptide may be provided as a fusion protein expressed from a chimeric gene in a host system lacking sterols, or translated in vitro using a cell-free system.
The steroylated-nucleic acid may comprise a canonical fused sterol or stanol ring system, a nucleophilic group at the 3-position of the A-ring of the fused sterol or stanol ring system, and an oligonucleotide attached through a linker to the fused sterol or stanol ring system.
The N-terminal peptide may have a length of between 2 amino acids and 500 amino acids.
The steroylated-nucleic acid may comprise at least one sterol molecule joined covalently to a nucleic acid polymer.
It is also an object to provide a composition, comprising: a polypeptide having a C-terminal glycine, a canonical fused sterol or stanol ring system, having a nucleophilic group at the 3-position of the A-ring of the fused sterol or stanol ring system, covalently linked to the C-terminal glycine, and an oligonucleotide attached through a linker adjacent to the sterol or stanol ring system.
The polypeptide may have between 2 and 500 amino acids.
The oligonucleotide may be an RNA, single stranded DNA, a segment of a double stranded DNA, or an oligonucleotide bound to an oligonucleotide strand having a complementary sequence.
The polypeptide and/or oligonucleotide may have a therapeutic activity in an animal, a diagnostic activity in an animal, a fluorescent property, and/or a fluorescence quenching property.
A therapeutic may be provided which selectively delivers a nucleotide or oligonucleotide to cells. The nucleotide may be effective to provide gene therapy, a DNA vaccine therapy, DNA nanostructure devices, etc. In this class of applications, the linked peptide serves to target and/or anchor the nucleotide in a desired region, tissue or cell type. On the other hand, in another class of applications, the nucleotide serves the purpose of anchoring or targeting, and the peptide provides a functional therapy or function. In other cases, both the peptide and the nucleotide may have specific targeting activities or functional activities. In a still further embodiment, the ligated peptide and nucleotide is itself linked or attached to a further active or targeting functionality. For example, if the peptide is an antigen, it may be complexed with a corresponding antibody. Similarly, the nucleotide may be annealed to a corresponding antisense nucleotide. Further, the peptide may have affinity for cofactors, and thus complex with the respective cofactor and carry that to a site of action. As party of a therapy, the active molecule, which may be the fusion peptide, or the fused product, is provided in a pharmaceutically acceptable dosage form, and administered to provide an effective therapy. The disease to be treated may be a neoplastic or hyperproliferative disease, an immune disorder, a genetic disorder, a disease to be treated with a gene therapy, miRNA, siRNA, dsRNA, or other oligonucleotide therapy.
For example, the polypeptide moiety may have receptor characteristics, or receptor-specific binding characteristics, to bind to a corresponding receptor or ligand on or in a specific tissue. The binding may lead to an endycytosis of the oligonucleotide, and thus obtain cell entry. The oligonucleotide may then act within the endocytosed environment, be released from the endosome into the cell, or the peptide-steroyl-oligonucleotide may act to lyse the endosome. In other cases, the peptide or oligonucleotide may act at the cell surface, external to the cell.
At least one of the polypeptide and the oligonucleotide may be bound to a support or a suspended particle.
The polypeptide may be configured to act as a sensor to report a presence and/or concentration of an analyte, as shown in
The polypeptide may be configured to act as an antigen for a corresponding antibody, or an antigen binding domain of an antibody.
The polypeptide may comprise a protease-sensitive domain, configured to release the oligonucleotide in response to cleavage of the protease-sensitive domain by a protease, as shown in
The polypeptide may comprise a receptor binding domain, configured to deliver the oligonucleotide to a specific cell type by receptor-mediated endocytosis, also shown in
These and other objects will become apparent from a review of the description herein.
According to one embodiment, the technology provides a method of conjugating a protein to a steroylated-oligonucleotide.
Chemical Synthesis of Steroylated-Oligonucleotide
A nominal 2-step protocol is provided to synthesize sterol-oligonucleotides via oxime chemistry, as shown schematically in
The protocol takes advantage of the fact that aminooxy groups react to form oximes in buffered aqueous conditions with equilibrium constants, typically in the range of >108 M−1.
Sterol-oligonucleotides compatible with HST-I catalyzed conjugation generally have the same general structure as (II) shown in
The reaction proceeds as follows:
Pregnenolone-16-ene oxime (PEG3) aminoxy (I): In a total volume of 1 ml consisting of 900 μl MeOH/100 μl triethanolamine acetate buffer (1 M, pH 7), dissolve 0.2 mmoles of Bis-(Aminooxy)-PEG3 with 0.1 mmoles of pregnenolone. Solution starts out cloudy, then turns clear after overnight mixing on a vortex. Oxime formation is monitored by TLC (95% Dichloromethane/5% methanol). Product, pregnonlone is isolated by organic extraction (3×) using 6 mls ethylacetate/6 mls water, followed by drying under nitrogen stream to a white solid. Typical yield is 70-85%.
Pregnenolone-16-ene bisoxime(PEG3)-oligo-dT20 (II): In microscale reaction, the product from step (I) is joined using the same oxime chemistry to an oligonucleotide equipped with a (4-formylbenzamide) group, obtained commercially (Solulink/TriLink Inc.). The solvent is 90/10, methanol/triethanolmamine acetate buffer (pH 7), with I at 0.02 M and the oligonucleotide 0.0002 M. Following overnight incubation, the sterol-oligonucleotide (II) is purified using microspin oligo clean and concentrator column (Zymogen Inc.), and eluted with water.
Fusion Protein
Create and clone a synthetic gene encoding POI fused to HST-I. The gene encoding the protein of interest (POI) is cloned into an expression plasmid, creating an in-frame translational fusion with HST-I. If the last amino acid of the target protein is not glycine, a glycine codon is added at the 3′ of the POI gene. This step involves standard molecular biology techniques.
Express POI-HST-I fusion protein. Recombinant vector encoding POI-HST-I fusion protein is transformed into a suitable expression host, e.g., E. coli, strain BL21 DE3.
Alternative hosts, ideally organisms that do not contain endogenous sterols, may be employed. Endogenous sterols may react with the POI-HST-I precursor protein, resulting in the release of POI.
It is also possible to produce the POI-HST-I fusion peptide by in vitro translation. See, www.neb.com/tools-and-resources/feature-articles/the-next-generation-of-cell-free-protein-synthesis; www.thermofisher.com/us/en/home/references/ambion-tech-support/large-scale-transcription/general-articles/the-basics-in-vitro-translation.html; en.wikipedia.org/wiki/Cell-free_protein_synthesis; Mikami S et al. (2006) A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins. J Biotechnol 127(1):65-78; Mikami S et al. (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46(2):348-57.
Purify POI-HST-1 fusion protein. Fusion protein is purified from the cell extract using an appropriate chromatography method. For example, immobilized metal affinity chromatography may be used (IMAC). Other purification techniques (GST-tag; chitin-tag; MBP-tag) could also be used.
Conjugation
Conjugation of POI to the sterol-oligonucleotide through the action of HST-I is initiated at room temperature by addition of a sterol-oligonucleotide to a final concentration 100-200 μM. Progress of the reaction can be followed by a variety of analytical methods. For example, SDS-PAGE may be used to monitor the change in molecular weight as the POI is conjugated and released from HST-1.
A final chromatography step can be carried out to separate HST-I from the conjugated target protein.
The feasibility of HST-I catalyzed conjugation of protein to nucleic acids has been assessed through pilot scale experiments. In one example, a chimeric gene encoding a 20 kDa POI fused to HST-1 was created. This gene product, a 46 kDa precursor polypeptide, was expressed in E. coli and purified under native conditions using immobilized metal affinity chromatography. To test conjugation activity, a 30 μl solution of the purified protein (2 μM, final) in BisTris buffered solution, was mixed with sterol-oligonucleotide (˜35 μM, final).
The oligonucleotide used in this experiment was chemically modified with a fluorescein group. After 3 hours at room temperature, contents of the reaction and control reactions were separated by SDS-PAGE. The gel was first imaged using UV light source to detect the fluorescent oligonucleotide, and then by Coomassie staining which detects all proteins, as shown in
Symbols:
In the sample containing the HST-I precursor protein, conjugation activity is indicated by the diminished staining of the precursor protein compared with control, as well as by the appearance of protein corresponding to the molecular weight of HST-I. Finally, in this sample, a high molecular weight product (dots) is observed that reacts with the Coomassie stain and gives off a fluorescence signal. Together, these characteristics indicate that this species is the desired protein-oligonucleotide conjugate.
Each reference cited herein is expressly incorporated herein by reference it its entirety.
It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification and the figures included herein. The embodiments, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.
The present application is a Division of U.S. patent application Ser. No. 15/782,391, filed Oct. 12, 2017, now U.S. Pat. No. 10,738,338, issued Aug. 11, 2020, which is a Non-provisional of, and claims benefit of priority from, U.S. Provisional Patent Application No. 62/409,655, filed Oct. 18, 2016, the entirety of which are expressly incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3773919 | Boswell et al. | Nov 1973 | A |
| 6544772 | Glenn et al. | Apr 2003 | B1 |
| 6620585 | Zyskind | Sep 2003 | B1 |
| 6773706 | Schneewind et al. | Aug 2004 | B2 |
| 6790448 | Xu et al. | Sep 2004 | B2 |
| 6838239 | Zyskind | Jan 2005 | B1 |
| 6841154 | Foster et al. | Jan 2005 | B2 |
| 6896887 | Leenhouts et al. | May 2005 | B2 |
| 6908994 | Rich et al. | Jun 2005 | B1 |
| 6936252 | Gilbert et al. | Aug 2005 | B2 |
| 6994982 | Watt et al. | Feb 2006 | B1 |
| 7067248 | Hruby et al. | Jun 2006 | B2 |
| 7067621 | Yeaman et al. | Jun 2006 | B2 |
| 7067639 | Eenhouts et al. | Jun 2006 | B2 |
| 7101692 | Schneewind et al. | Sep 2006 | B2 |
| 7125698 | Glenn et al. | Oct 2006 | B2 |
| 7179458 | Chang et al. | Feb 2007 | B2 |
| 7183101 | Arigoni et al. | Feb 2007 | B2 |
| 7195763 | Xu et al. | Mar 2007 | B2 |
| 7238489 | Schneewind et al. | Jul 2007 | B2 |
| 7270969 | Watt et al. | Sep 2007 | B2 |
| 7312076 | Chang et al. | Dec 2007 | B2 |
| 7348420 | Klaenhammer et al. | Mar 2008 | B2 |
| 7371719 | Stupp et al. | May 2008 | B2 |
| 7384775 | Zagursky et al. | Jun 2008 | B2 |
| 7390526 | Stupp et al. | Jun 2008 | B2 |
| 7452679 | Stupp et al. | Nov 2008 | B2 |
| 7455992 | Klaenhammer et al. | Nov 2008 | B2 |
| 7456011 | Liu et al. | Nov 2008 | B2 |
| 7485710 | Reinscheid et al. | Feb 2009 | B2 |
| 7491690 | Stupp et al. | Feb 2009 | B2 |
| 7534761 | Stupp et al. | May 2009 | B1 |
| 7534876 | Klaenhammer et al. | May 2009 | B2 |
| 7538209 | Klaenhammer et al. | May 2009 | B2 |
| 7541039 | Leenhouts et al. | Jun 2009 | B2 |
| 7544661 | Stupp et al. | Jun 2009 | B2 |
| 7554021 | Stupp et al. | Jun 2009 | B2 |
| 7615616 | Hook et al. | Nov 2009 | B2 |
| 7632515 | Gilbert et al. | Dec 2009 | B2 |
| 7635487 | Meinke et al. | Dec 2009 | B2 |
| 7648708 | Gilbert et al. | Jan 2010 | B2 |
| 7683025 | Stupp et al. | Mar 2010 | B2 |
| 7709009 | Grandi et al. | May 2010 | B2 |
| 7713534 | Gilbert et al. | May 2010 | B2 |
| 7722888 | Gilbert et al. | May 2010 | B2 |
| 7731978 | Bensi et al. | Jun 2010 | B2 |
| 7745708 | Stupp et al. | Jun 2010 | B2 |
| 7754467 | Chang et al. | Jul 2010 | B2 |
| 7763420 | Stritzker et al. | Jul 2010 | B2 |
| 7771728 | Meinke et al. | Aug 2010 | B2 |
| 7771731 | Matsuka et al. | Aug 2010 | B2 |
| 7776553 | Love et al. | Aug 2010 | B2 |
| 7776589 | Prongay et al. | Aug 2010 | B1 |
| 7785608 | Zlotnick et al. | Aug 2010 | B2 |
| 7803765 | Watt et al. | Sep 2010 | B2 |
| 7820184 | Stritzker et al. | Oct 2010 | B2 |
| 7829681 | Seefeldt et al. | Nov 2010 | B2 |
| 7833791 | Chang et al. | Nov 2010 | B2 |
| 7838010 | Bensi et al. | Nov 2010 | B2 |
| 7838491 | Stupp et al. | Nov 2010 | B2 |
| 7850974 | Hook et al. | Dec 2010 | B2 |
| 7851445 | Stupp et al. | Dec 2010 | B2 |
| 7858357 | Leenhouts et al. | Dec 2010 | B2 |
| 7888075 | McCarthy et al. | Feb 2011 | B2 |
| 7897367 | Klaenhammer et al. | Mar 2011 | B2 |
| 7927864 | Owen | Apr 2011 | B2 |
| 7935804 | Dubensky, Jr. et al. | May 2011 | B2 |
| 7939087 | Telford et al. | May 2011 | B2 |
| 7955604 | Telford et al. | Jun 2011 | B2 |
| 7960505 | OSullivan et al. | Jun 2011 | B2 |
| 7960533 | Reinscheid et al. | Jun 2011 | B2 |
| 7968100 | Foster et al. | Jun 2011 | B2 |
| 7968297 | Meinke et al. | Jun 2011 | B2 |
| 7968683 | Beyer et al. | Jun 2011 | B1 |
| 8007803 | Emery et al. | Aug 2011 | B2 |
| 8007811 | Emery et al. | Aug 2011 | B2 |
| 8025885 | Emery et al. | Sep 2011 | B2 |
| 8025890 | Telford et al. | Sep 2011 | B2 |
| 8038990 | Wang et al. | Oct 2011 | B2 |
| 8039005 | Bensi et al. | Oct 2011 | B2 |
| 8063014 | Stupp et al. | Nov 2011 | B2 |
| 8076295 | Hulvat et al. | Dec 2011 | B2 |
| 8088611 | Prongay et al. | Jan 2012 | B2 |
| 8101194 | Zlotnick et al. | Jan 2012 | B2 |
| 8105612 | Soriani et al. | Jan 2012 | B2 |
| 8110199 | Gilbert et al. | Feb 2012 | B2 |
| 8124107 | Hook et al. | Feb 2012 | B2 |
| 8124583 | Stupp et al. | Feb 2012 | B2 |
| 8128936 | Grandi et al. | Mar 2012 | B2 |
| 8137673 | Telford et al. | Mar 2012 | B2 |
| 8138140 | Stupp et al. | Mar 2012 | B2 |
| 8148321 | Roy et al. | Apr 2012 | B2 |
| 8241642 | Zagursky et al. | Aug 2012 | B2 |
| 8252546 | Briles et al. | Aug 2012 | B2 |
| 8263642 | Skaar et al. | Sep 2012 | B2 |
| 8280643 | Hook et al. | Oct 2012 | B2 |
| 8287885 | Margarit Y Ros et al. | Oct 2012 | B2 |
| 8318908 | Reinscheid et al. | Nov 2012 | B2 |
| 8323660 | Meinke et al. | Dec 2012 | B2 |
| 8329195 | Briles et al. | Dec 2012 | B2 |
| 8372411 | Meinke et al. | Feb 2013 | B2 |
| 8377446 | Soriani et al. | Feb 2013 | B2 |
| 8399651 | Margarit Y Ros et al. | Mar 2013 | B2 |
| 8409589 | Bensi et al. | Apr 2013 | B2 |
| 8431139 | Telford et al. | Apr 2013 | B2 |
| 8445426 | De Vos et al. | May 2013 | B2 |
| 8450271 | Shah et al. | May 2013 | B2 |
| 8475809 | Leigh | Jul 2013 | B2 |
| 8524241 | Seed et al. | Sep 2013 | B2 |
| 8529913 | Grandi et al. | Sep 2013 | B2 |
| 8557961 | Silverman et al. | Oct 2013 | B2 |
| 8563001 | Dodge et al. | Oct 2013 | B2 |
| 8563006 | Zlotnick et al. | Oct 2013 | B2 |
| 8563007 | Zlotnick et al. | Oct 2013 | B1 |
| 8568735 | Anderson et al. | Oct 2013 | B2 |
| 8574597 | Zlotnick | Nov 2013 | B2 |
| 8575070 | Watt et al. | Nov 2013 | B2 |
| 8580923 | Stupp et al. | Nov 2013 | B2 |
| 8580939 | Dubensky, Jr. et al. | Nov 2013 | B2 |
| 8591899 | Shafferman et al. | Nov 2013 | B2 |
| 8592375 | Yeaman et al. | Nov 2013 | B2 |
| 8598342 | Kahne et al. | Dec 2013 | B2 |
| 8609106 | Masignani et al. | Dec 2013 | B2 |
| 8617556 | Beaumont et al. | Dec 2013 | B2 |
| 8632783 | Bagnoli et al. | Jan 2014 | B2 |
| 8647835 | Walsh et al. | Feb 2014 | B2 |
| 8652800 | Walsh et al. | Feb 2014 | B2 |
| 8663631 | Quinn | Mar 2014 | B2 |
| 8663926 | Boyer et al. | Mar 2014 | B2 |
| 8669226 | Bond et al. | Mar 2014 | B2 |
| 8673860 | Schellenberger et al. | Mar 2014 | B2 |
| 8679505 | Bagnoli et al. | Mar 2014 | B2 |
| 8680050 | Schellenberger et al. | Mar 2014 | B2 |
| 8703717 | Schellenberger et al. | Apr 2014 | B2 |
| 8709431 | Chowdari et al. | Apr 2014 | B2 |
| 8709436 | Emery et al. | Apr 2014 | B2 |
| 8709760 | Emery et al. | Apr 2014 | B2 |
| 8710188 | Beyer et al. | Apr 2014 | B2 |
| 8715688 | Meinke et al. | May 2014 | B2 |
| 8716448 | Schellenberger et al. | May 2014 | B2 |
| 8722354 | Sjong et al. | May 2014 | B2 |
| 8748122 | Hyman et al. | Jun 2014 | B2 |
| 8754198 | Lunder et al. | Jun 2014 | B2 |
| 8758765 | Missiakas et al. | Jun 2014 | B2 |
| 8772049 | Love et al. | Jul 2014 | B2 |
| 8778358 | Telford et al. | Jul 2014 | B2 |
| 8795965 | Zhang | Aug 2014 | B2 |
| 8795983 | Hyman et al. | Aug 2014 | B2 |
| 8808699 | Schneewind et al. | Aug 2014 | B2 |
| 8821894 | Schneewind et al. | Sep 2014 | B2 |
| 8822409 | Milech et al. | Sep 2014 | B2 |
| 8835187 | Love et al. | Sep 2014 | B2 |
| 8835188 | Love et al. | Sep 2014 | B2 |
| 8840906 | Bubeck-Wardenburg et al. | Sep 2014 | B2 |
| 8841249 | Johansen et al. | Sep 2014 | B2 |
| 8853382 | Hammarstrom et al. | Oct 2014 | B2 |
| 8858957 | Margarit Y Ros et al. | Oct 2014 | B2 |
| 8859492 | Cowan et al. | Oct 2014 | B2 |
| 8865479 | Love et al. | Oct 2014 | B2 |
| 8871204 | Brezski et al. | Oct 2014 | B2 |
| 8871445 | Cong et al. | Oct 2014 | B2 |
| 8883788 | Hasui et al. | Nov 2014 | B2 |
| 8889145 | Anderson et al. | Nov 2014 | B2 |
| 8889150 | Malouin et al. | Nov 2014 | B2 |
| 8889356 | Zhang | Nov 2014 | B2 |
| 8927230 | Hoess et al. | Jan 2015 | B2 |
| 8932814 | Cong et al. | Jan 2015 | B2 |
| 8933193 | OSullivan et al. | Jan 2015 | B2 |
| 8933197 | Stemmer et al. | Jan 2015 | B2 |
| 8937167 | Janetka et al. | Jan 2015 | B2 |
| 8940501 | Ploegh et al. | Jan 2015 | B2 |
| 8945542 | Heartlein et al. | Feb 2015 | B2 |
| 8945588 | Schneewind et al. | Feb 2015 | B2 |
| 8945589 | Telford et al. | Feb 2015 | B2 |
| 8945855 | Iverson et al. | Feb 2015 | B2 |
| 8946381 | Fear et al. | Feb 2015 | B2 |
| 8957021 | Schellenberger et al. | Feb 2015 | B2 |
| 8961979 | Emery et al. | Feb 2015 | B2 |
| 8975232 | Liu et al. | Mar 2015 | B2 |
| 8980284 | Ichtchenko et al. | Mar 2015 | B2 |
| 8980824 | Cong et al. | Mar 2015 | B2 |
| 8986710 | Anderson et al. | Mar 2015 | B2 |
| 8993295 | Seed et al. | Mar 2015 | B2 |
| 9005579 | Nowinski et al. | Apr 2015 | B2 |
| 9050374 | Watts et al. | Jun 2015 | B2 |
| 9056912 | Grandi et al. | Jun 2015 | B2 |
| 9062299 | Schellenberger et al. | Jun 2015 | B2 |
| 9068985 | Sjong et al. | Jun 2015 | B2 |
| 9079946 | Grandi et al. | Jul 2015 | B2 |
| 9079952 | Collier et al. | Jul 2015 | B2 |
| 9080159 | Briles et al. | Jul 2015 | B2 |
| 9090677 | Beaumont et al. | Jul 2015 | B2 |
| 9095540 | Schneewind et al. | Aug 2015 | B2 |
| 9102741 | Margarit Y Ros et al. | Aug 2015 | B2 |
| 9107873 | Zlotnick et al. | Aug 2015 | B2 |
| 9109008 | Cong et al. | Aug 2015 | B2 |
| 9114105 | Anderson et al. | Aug 2015 | B2 |
| 9127050 | Scully et al. | Sep 2015 | B2 |
| 9128058 | Walsh et al. | Sep 2015 | B2 |
| 9129785 | Dulay et al. | Sep 2015 | B2 |
| 9132179 | Van Ginkel et al. | Sep 2015 | B2 |
| 9132182 | Zlotnick et al. | Sep 2015 | B2 |
| 9134304 | Wagner et al. | Sep 2015 | B2 |
| 9150626 | Liu et al. | Oct 2015 | B2 |
| 9156850 | Chowdari et al. | Oct 2015 | B2 |
| 9168293 | Zlotnick et al. | Oct 2015 | B2 |
| 9168312 | Schellenberger et al. | Oct 2015 | B2 |
| 9181297 | Pentelute et al. | Nov 2015 | B1 |
| 9181329 | Bubeck-Wardenburg et al. | Nov 2015 | B2 |
| 9182390 | Nishimura et al. | Nov 2015 | B2 |
| 9205142 | Bagnoli et al. | Dec 2015 | B2 |
| 9212219 | Schneewind et al. | Dec 2015 | B2 |
| 9217157 | Garcia-Sastre et al. | Dec 2015 | B2 |
| 9221882 | Skerra et al. | Dec 2015 | B2 |
| 9221886 | Liu et al. | Dec 2015 | B2 |
| 9221902 | Smider et al. | Dec 2015 | B2 |
| 9234012 | Saito et al. | Jan 2016 | B2 |
| 9238010 | Hammer et al. | Jan 2016 | B2 |
| 9243038 | Liu et al. | Jan 2016 | B2 |
| 9249211 | Schellenberger et al. | Feb 2016 | B2 |
| 9266925 | Zecri et al. | Feb 2016 | B2 |
| 9266943 | Beaumont et al. | Feb 2016 | B2 |
| 9266944 | Emery et al. | Feb 2016 | B1 |
| 9267127 | Liu et al. | Feb 2016 | B2 |
| 9315554 | Schneewind et al. | Apr 2016 | B2 |
| 9340582 | Yuan et al. | May 2016 | B2 |
| 9340584 | Wolfe et al. | May 2016 | B2 |
| 9353160 | Foster et al. | May 2016 | B2 |
| 9371369 | Schellenberger et al. | Jun 2016 | B2 |
| 9376672 | Schellenberger et al. | Jun 2016 | B2 |
| 9382289 | Cong et al. | Jul 2016 | B2 |
| 9388225 | Del Campo Ascarateil et al. | Jul 2016 | B2 |
| 9393294 | Gierahn et al. | Jul 2016 | B2 |
| 9394092 | Lee et al. | Jul 2016 | B2 |
| 9399673 | Beaumont et al. | Jul 2016 | B2 |
| 9403904 | Smider et al. | Aug 2016 | B2 |
| 9404922 | Fischetti et al. | Aug 2016 | B2 |
| 9405069 | Fattinger | Aug 2016 | B2 |
| 9408890 | Comolli et al. | Aug 2016 | B2 |
| 9409952 | Kariyuki et al. | Aug 2016 | B2 |
| 9416171 | Lydon | Aug 2016 | B2 |
| 9434774 | Liu et al. | Sep 2016 | B2 |
| 9441016 | Altermann et al. | Sep 2016 | B2 |
| 9458228 | Beaumont et al. | Oct 2016 | B2 |
| 9458229 | Grandi et al. | Oct 2016 | B2 |
| 9463431 | Love et al. | Oct 2016 | B2 |
| 10100080 | Pallisse Bergwerf | Oct 2018 | B2 |
| 20020028457 | Empedocles et al. | Mar 2002 | A1 |
| 20030021789 | Xu et al. | Jan 2003 | A1 |
| 20030022178 | Schneewind et al. | Jan 2003 | A1 |
| 20030087864 | Talbot et al. | May 2003 | A1 |
| 20030091577 | Gilbert et al. | May 2003 | A1 |
| 20030099940 | Empedocles et al. | May 2003 | A1 |
| 20030153020 | Schneewind et al. | Aug 2003 | A1 |
| 20030180816 | Leenhouts et al. | Sep 2003 | A1 |
| 20030185833 | Foster et al. | Oct 2003 | A1 |
| 20030186851 | Leenhouts et al. | Oct 2003 | A1 |
| 20030228297 | Chang et al. | Dec 2003 | A1 |
| 20040091856 | Pelletier et al. | May 2004 | A1 |
| 20040101919 | Hook et al. | May 2004 | A1 |
| 20040110181 | Zagursky et al. | Jun 2004 | A1 |
| 20040126870 | Arigoni et al. | Jul 2004 | A1 |
| 20040167068 | Zlotnick et al. | Aug 2004 | A1 |
| 20040230033 | Walker | Nov 2004 | A1 |
| 20040236072 | Olmsted et al. | Nov 2004 | A1 |
| 20050002925 | Xu et al. | Jan 2005 | A1 |
| 20050003510 | Chang et al. | Jan 2005 | A1 |
| 20050037444 | Meinke et al. | Feb 2005 | A1 |
| 20050048545 | Cull et al. | Mar 2005 | A1 |
| 20050069984 | Schneewind et al. | Mar 2005 | A1 |
| 20050106648 | Foster et al. | May 2005 | A1 |
| 20050112612 | Klaenhammer et al. | May 2005 | A1 |
| 20050175581 | Haupts et al. | Aug 2005 | A1 |
| 20050203280 | McMichael et al. | Sep 2005 | A1 |
| 20050207995 | Gregory et al. | Sep 2005 | A1 |
| 20050220788 | Nagy et al. | Oct 2005 | A1 |
| 20050233396 | Hruby et al. | Oct 2005 | A1 |
| 20050276814 | Gilbert et al. | Dec 2005 | A1 |
| 20060073530 | Schneewind et al. | Apr 2006 | A1 |
| 20060074016 | Yeaman et al. | Apr 2006 | A1 |
| 20060078901 | Buchrieser et al. | Apr 2006 | A1 |
| 20060115491 | Leenhouts et al. | Jun 2006 | A1 |
| 20060135416 | Yeaman et al. | Jun 2006 | A1 |
| 20060165716 | Telford et al. | Jul 2006 | A1 |
| 20060177462 | Anderson et al. | Aug 2006 | A1 |
| 20060188975 | Ramaswami | Aug 2006 | A1 |
| 20060194226 | Liu et al. | Aug 2006 | A1 |
| 20060198852 | Hook et al. | Sep 2006 | A1 |
| 20060234222 | McKeown et al. | Oct 2006 | A1 |
| 20060246080 | Alibek et al. | Nov 2006 | A1 |
| 20060257413 | Zlotnick et al. | Nov 2006 | A1 |
| 20060263846 | Meinke et al. | Nov 2006 | A1 |
| 20060269538 | Koltermann et al. | Nov 2006 | A1 |
| 20060275315 | Telford et al. | Dec 2006 | A1 |
| 20070003667 | Klaenhammer et al. | Jan 2007 | A1 |
| 20070026011 | Liu et al. | Feb 2007 | A1 |
| 20070031832 | Watt et al. | Feb 2007 | A1 |
| 20070053924 | Tettelin et al. | Mar 2007 | A1 |
| 20070059295 | Wang et al. | Mar 2007 | A1 |
| 20070082006 | Zlotnick et al. | Apr 2007 | A1 |
| 20070082007 | Zlotnick et al. | Apr 2007 | A1 |
| 20070082866 | Zlotnick et al. | Apr 2007 | A1 |
| 20070117197 | Chang et al. | May 2007 | A1 |
| 20070128210 | Olmsted et al. | Jun 2007 | A1 |
| 20070128211 | Olmsted et al. | Jun 2007 | A1 |
| 20070128229 | Olmsted et al. | Jun 2007 | A1 |
| 20070172495 | Klaenhammer et al. | Jul 2007 | A1 |
| 20070190029 | Pardoll et al. | Aug 2007 | A1 |
| 20070190063 | Bahjat et al. | Aug 2007 | A1 |
| 20070207170 | Dubensky et al. | Sep 2007 | A1 |
| 20070207171 | Dubensky et al. | Sep 2007 | A1 |
| 20070218075 | Matsuka et al. | Sep 2007 | A1 |
| 20070248581 | Chen et al. | Oct 2007 | A1 |
| 20070253964 | Zlotnick et al. | Nov 2007 | A1 |
| 20070258955 | Klaenhammer et al. | Nov 2007 | A1 |
| 20070286866 | Van Ginkel et al. | Dec 2007 | A1 |
| 20080031877 | Covacci et al. | Feb 2008 | A1 |
| 20080038287 | Meinke et al. | Feb 2008 | A1 |
| 20080050361 | Heinrichs et al. | Feb 2008 | A1 |
| 20080064079 | Hoess et al. | Mar 2008 | A1 |
| 20080089899 | Gilbert et al. | Apr 2008 | A1 |
| 20080171059 | Howland et al. | Jul 2008 | A1 |
| 20080175857 | Gilbert et al. | Jul 2008 | A1 |
| 20080220441 | Birnbaum et al. | Sep 2008 | A1 |
| 20080248522 | Seefeldt et al. | Oct 2008 | A1 |
| 20080254070 | Gilbert et al. | Oct 2008 | A1 |
| 20080260768 | Gilbert et al. | Oct 2008 | A1 |
| 20090022753 | Olmsted et al. | Jan 2009 | A1 |
| 20090035780 | McCarthy et al. | Feb 2009 | A1 |
| 20090041744 | Ostergaard et al. | Feb 2009 | A1 |
| 20090075839 | Zagursky et al. | Mar 2009 | A1 |
| 20090088337 | Gill et al. | Apr 2009 | A1 |
| 20090088372 | Roy et al. | Apr 2009 | A1 |
| 20090092582 | Bogin et al. | Apr 2009 | A1 |
| 20090117113 | Bensi et al. | May 2009 | A1 |
| 20090130115 | Hook et al. | May 2009 | A1 |
| 20090148408 | Chang et al. | Jun 2009 | A1 |
| 20090155304 | Liu et al. | Jun 2009 | A1 |
| 20090176967 | Stennicke | Jul 2009 | A1 |
| 20090202578 | Foster et al. | Aug 2009 | A1 |
| 20090202593 | Zlotnick et al. | Aug 2009 | A1 |
| 20090214476 | Pretzer et al. | Aug 2009 | A1 |
| 20090214537 | Soriani et al. | Aug 2009 | A1 |
| 20090214584 | Guss et al. | Aug 2009 | A1 |
| 20090239264 | Leenhouts et al. | Sep 2009 | A1 |
| 20090297548 | Kudva et al. | Dec 2009 | A1 |
| 20090297549 | Tettelin et al. | Dec 2009 | A1 |
| 20090305252 | Li et al. | Dec 2009 | A1 |
| 20090317420 | Telford et al. | Dec 2009 | A1 |
| 20090317421 | Missiakas et al. | Dec 2009 | A1 |
| 20100004324 | Skaar et al. | Jan 2010 | A1 |
| 20100009917 | Buchardt et al. | Jan 2010 | A1 |
| 20100055761 | Seed et al. | Mar 2010 | A1 |
| 20100064393 | Berka et al. | Mar 2010 | A1 |
| 20100068220 | Hook et al. | Mar 2010 | A1 |
| 20100074923 | Covacci et al. | Mar 2010 | A1 |
| 20100098789 | Balambika et al. | Apr 2010 | A1 |
| 20100105865 | Telford et al. | Apr 2010 | A1 |
| 20100119534 | Dodge et al. | May 2010 | A1 |
| 20100150943 | Grandi et al. | Jun 2010 | A1 |
| 20100152054 | Love et al. | Jun 2010 | A1 |
| 20100183614 | Paul et al. | Jul 2010 | A1 |
| 20100184624 | Samuel et al. | Jul 2010 | A1 |
| 20100196524 | Meindert De Vos et al. | Aug 2010 | A1 |
| 20100221288 | Zagursky et al. | Sep 2010 | A1 |
| 20100227341 | Briles et al. | Sep 2010 | A1 |
| 20100239554 | Schellenberger et al. | Sep 2010 | A1 |
| 20100247561 | Anderson et al. | Sep 2010 | A1 |
| 20100255026 | Stump et al. | Oct 2010 | A1 |
| 20100256070 | Seed et al. | Oct 2010 | A1 |
| 20100260706 | Bogin et al. | Oct 2010 | A1 |
| 20100260790 | Meinke et al. | Oct 2010 | A1 |
| 20100267053 | Prongay et al. | Oct 2010 | A1 |
| 20100278740 | Gilbert et al. | Nov 2010 | A1 |
| 20100279328 | Hong et al. | Nov 2010 | A1 |
| 20100297183 | Smith | Nov 2010 | A1 |
| 20100303864 | Tettelin et al. | Dec 2010 | A1 |
| 20100323956 | Schellenberger et al. | Dec 2010 | A1 |
| 20110020323 | Beaumont et al. | Jan 2011 | A1 |
| 20110020385 | Masignani et al. | Jan 2011 | A1 |
| 20110020402 | Meinke et al. | Jan 2011 | A1 |
| 20110020900 | Klaenhammer et al. | Jan 2011 | A1 |
| 20110046008 | Love et al. | Feb 2011 | A1 |
| 20110046060 | Schellenberger et al. | Feb 2011 | A1 |
| 20110046061 | Schellenberger et al. | Feb 2011 | A1 |
| 20110076299 | Zlotnick et al. | Mar 2011 | A1 |
| 20110077199 | Schellenberger et al. | Mar 2011 | A1 |
| 20110091956 | Nishimura et al. | Apr 2011 | A1 |
| 20110097360 | Donati et al. | Apr 2011 | A1 |
| 20110104168 | Briles et al. | May 2011 | A1 |
| 20110110982 | Telford et al. | May 2011 | A1 |
| 20110124520 | Love et al. | May 2011 | A1 |
| 20110129935 | Schaeffer | Jun 2011 | A1 |
| 20110150918 | Foster et al. | Jun 2011 | A1 |
| 20110151053 | Klaenhammer et al. | Jun 2011 | A1 |
| 20110172146 | Schellenberger et al. | Jul 2011 | A1 |
| 20110177976 | Gordon et al. | Jul 2011 | A1 |
| 20110183863 | Wagner et al. | Jul 2011 | A1 |
| 20110189187 | Zlotnick | Aug 2011 | A1 |
| 20110189236 | Scott et al. | Aug 2011 | A1 |
| 20110189664 | Dixon et al. | Aug 2011 | A1 |
| 20110206616 | Ichtchenko et al. | Aug 2011 | A1 |
| 20110206676 | Missiakas et al. | Aug 2011 | A1 |
| 20110206692 | Maione et al. | Aug 2011 | A1 |
| 20110243976 | Donati et al. | Oct 2011 | A1 |
| 20110243977 | Olmsted et al. | Oct 2011 | A1 |
| 20110245480 | Dubensky, Jr. et al. | Oct 2011 | A1 |
| 20110262477 | Cheng et al. | Oct 2011 | A1 |
| 20110263501 | Johansen | Oct 2011 | A1 |
| 20110275132 | Covacci et al. | Nov 2011 | A1 |
| 20110281745 | Love et al. | Nov 2011 | A1 |
| 20110281764 | Love et al. | Nov 2011 | A1 |
| 20110288005 | Silverman et al. | Nov 2011 | A1 |
| 20110311536 | Von Boehmer et al. | Dec 2011 | A1 |
| 20110312881 | Silverman et al. | Dec 2011 | A1 |
| 20110318339 | Smider et al. | Dec 2011 | A1 |
| 20110321183 | Ploegh et al. | Dec 2011 | A1 |
| 20120015379 | Shafferman et al. | Jan 2012 | A1 |
| 20120034261 | Zlotnick et al. | Feb 2012 | A1 |
| 20120058906 | Smider et al. | Mar 2012 | A1 |
| 20120064103 | Giuliani et al. | Mar 2012 | A1 |
| 20120064104 | Costantino | Mar 2012 | A1 |
| 20120076814 | Masignani et al. | Mar 2012 | A1 |
| 20120083599 | Thomas et al. | Apr 2012 | A1 |
| 20120093840 | Ostergaard et al. | Apr 2012 | A1 |
| 20120093850 | Bagnoli et al. | Apr 2012 | A1 |
| 20120100174 | Leigh | Apr 2012 | A1 |
| 20120100569 | Liu et al. | Apr 2012 | A1 |
| 20120107340 | Bagnoli et al. | May 2012 | A1 |
| 20120114686 | Schneewind et al. | May 2012 | A1 |
| 20120121643 | Dubensky, Jr. et al. | May 2012 | A1 |
| 20120122123 | Boyer et al. | May 2012 | A1 |
| 20120142682 | Merrill | Jun 2012 | A1 |
| 20120149590 | Klaenhammer et al. | Jun 2012 | A1 |
| 20120149710 | Jung et al. | Jun 2012 | A1 |
| 20120157665 | Beaumont et al. | Jun 2012 | A1 |
| 20120171211 | Soriani et al. | Jul 2012 | A1 |
| 20120172303 | Johansen et al. | Jul 2012 | A1 |
| 20120178691 | Schellenberger et al. | Jul 2012 | A1 |
| 20120189649 | Gierahn et al. | Jul 2012 | A1 |
| 20120201844 | Zlotnick et al. | Aug 2012 | A1 |
| 20120207778 | Telford et al. | Aug 2012 | A1 |
| 20120237536 | Rappuoli et al. | Sep 2012 | A1 |
| 20120244189 | Foster et al. | Sep 2012 | A1 |
| 20120251568 | Garcia-Sastre et al. | Oct 2012 | A1 |
| 20120263701 | Schellenberger et al. | Oct 2012 | A1 |
| 20120263703 | Schellenberger et al. | Oct 2012 | A1 |
| 20120270797 | Wittrup et al. | Oct 2012 | A1 |
| 20120282247 | Schneewind et al. | Nov 2012 | A1 |
| 20120282289 | Bannoehr et al. | Nov 2012 | A1 |
| 20120282670 | Rossomando | Nov 2012 | A1 |
| 20120282700 | Lunder et al. | Nov 2012 | A1 |
| 20120289454 | Cowan et al. | Nov 2012 | A1 |
| 20120294880 | Zlotnick et al. | Nov 2012 | A1 |
| 20120301428 | Wren et al. | Nov 2012 | A1 |
| 20120301433 | Lu et al. | Nov 2012 | A1 |
| 20120301496 | Zlotnick et al. | Nov 2012 | A1 |
| 20120308595 | Zlotnick et al. | Dec 2012 | A1 |
| 20120309679 | Hesse et al. | Dec 2012 | A1 |
| 20120309701 | Janetka et al. | Dec 2012 | A1 |
| 20120316071 | Smider et al. | Dec 2012 | A1 |
| 20130011386 | Brezski et al. | Jan 2013 | A1 |
| 20130011428 | Zagursky et al. | Jan 2013 | A1 |
| 20130017997 | Schellenberger et al. | Jan 2013 | A1 |
| 20130034575 | Meinke et al. | Feb 2013 | A1 |
| 20130034847 | Kojic et al. | Feb 2013 | A1 |
| 20130039884 | Bogin et al. | Feb 2013 | A1 |
| 20130045211 | Nowinski et al. | Feb 2013 | A1 |
| 20130064845 | Malouin et al. | Mar 2013 | A1 |
| 20130071416 | Grandi et al. | Mar 2013 | A1 |
| 20130072420 | Skerra et al. | Mar 2013 | A1 |
| 20130084648 | Bolton et al. | Apr 2013 | A1 |
| 20130089525 | Bond et al. | Apr 2013 | A1 |
| 20130101665 | Ugolin et al. | Apr 2013 | A1 |
| 20130122043 | Guimaraes et al. | May 2013 | A1 |
| 20130136746 | Schneewind et al. | May 2013 | A1 |
| 20130136761 | Meinke et al. | May 2013 | A1 |
| 20130143955 | Breaker et al. | Jun 2013 | A1 |
| 20130157281 | Beyer et al. | Jun 2013 | A1 |
| 20130165389 | Schellenberger et al. | Jun 2013 | A1 |
| 20130171183 | Schneewind | Jul 2013 | A1 |
| 20130177940 | Hoess et al. | Jul 2013 | A1 |
| 20130184177 | Bosma | Jul 2013 | A1 |
| 20130189287 | Bregeon et al. | Jul 2013 | A1 |
| 20130209494 | Chowdari et al. | Aug 2013 | A1 |
| 20130216568 | Maione et al. | Aug 2013 | A1 |
| 20130217592 | Samuel et al. | Aug 2013 | A1 |
| 20130217612 | Altermann et al. | Aug 2013 | A1 |
| 20130230550 | Schneewind et al. | Sep 2013 | A1 |
| 20130236419 | Schneewind et al. | Sep 2013 | A1 |
| 20130243818 | Leigh | Sep 2013 | A1 |
| 20130253175 | Beaumont et al. | Sep 2013 | A1 |
| 20130259889 | Zlotnick et al. | Oct 2013 | A1 |
| 20130260404 | Sjong et al. | Oct 2013 | A1 |
| 20130261293 | Beaumont et al. | Oct 2013 | A1 |
| 20130288266 | Gerg et al. | Oct 2013 | A1 |
| 20130288267 | Gerg et al. | Oct 2013 | A1 |
| 20130289251 | Gallusser et al. | Oct 2013 | A1 |
| 20130289253 | Uescher et al. | Oct 2013 | A1 |
| 20130296257 | Saito et al. | Nov 2013 | A1 |
| 20130316946 | Barrack | Nov 2013 | A1 |
| 20130323819 | Hammarstrom et al. | Dec 2013 | A1 |
| 20130330335 | Bremel et al. | Dec 2013 | A1 |
| 20130338030 | Love et al. | Dec 2013 | A1 |
| 20130338047 | Love et al. | Dec 2013 | A1 |
| 20130344010 | Pompejus | Dec 2013 | A1 |
| 20140004138 | Briles et al. | Jan 2014 | A1 |
| 20140011709 | Love et al. | Jan 2014 | A1 |
| 20140017764 | Iverson et al. | Jan 2014 | A1 |
| 20140030697 | Ploegh et al. | Jan 2014 | A1 |
| 20140037650 | Kim et al. | Feb 2014 | A1 |
| 20140037669 | Scully et al. | Feb 2014 | A1 |
| 20140051834 | Hoffman et al. | Feb 2014 | A1 |
| 20140057317 | Liu et al. | Feb 2014 | A1 |
| 20140065171 | Geierstanger et al. | Mar 2014 | A1 |
| 20140073639 | Fischetti et al. | Mar 2014 | A1 |
| 20140105818 | Hammer et al. | Apr 2014 | A1 |
| 20140113832 | Wolfe et al. | Apr 2014 | A1 |
| 20140128289 | Gordon et al. | May 2014 | A1 |
| 20140147873 | Clubb et al. | May 2014 | A1 |
| 20140154286 | Malley et al. | Jun 2014 | A1 |
| 20140154287 | Malley et al. | Jun 2014 | A1 |
| 20140155319 | Bond et al. | Jun 2014 | A1 |
| 20140161915 | Payne et al. | Jun 2014 | A1 |
| 20140162949 | Cleland et al. | Jun 2014 | A1 |
| 20140170702 | Reitmeir et al. | Jun 2014 | A1 |
| 20140178425 | Bagnoli et al. | Jun 2014 | A1 |
| 20140179006 | Zhang | Jun 2014 | A1 |
| 20140186265 | McNaughton et al. | Jul 2014 | A1 |
| 20140186327 | Schellenberger et al. | Jul 2014 | A1 |
| 20140186350 | Ghosh et al. | Jul 2014 | A1 |
| 20140186354 | Bossenmaier et al. | Jul 2014 | A1 |
| 20140186358 | Bossenmaier et al. | Jul 2014 | A1 |
| 20140189896 | Zhang et al. | Jul 2014 | A1 |
| 20140193438 | Chowdari et al. | Jul 2014 | A1 |
| 20140206840 | Sjong et al. | Jul 2014 | A1 |
| 20140213515 | Liu et al. | Jul 2014 | A1 |
| 20140227295 | Cong et al. | Aug 2014 | A1 |
| 20140227298 | Cong et al. | Aug 2014 | A1 |
| 20140234972 | Zhang | Aug 2014 | A1 |
| 20140235828 | Beaumont et al. | Aug 2014 | A1 |
| 20140243280 | Herrmann et al. | Aug 2014 | A1 |
| 20140248702 | Zhang et al. | Sep 2014 | A1 |
| 20140249296 | Ploegh et al. | Sep 2014 | A1 |
| 20140255470 | Comolli et al. | Sep 2014 | A1 |
| 20140273231 | Zhang et al. | Sep 2014 | A1 |
| 20140287509 | Sharei et al. | Sep 2014 | A1 |
| 20140301974 | Schellenberger et al. | Oct 2014 | A1 |
| 20140302084 | Schneewind et al. | Oct 2014 | A1 |
| 20140308318 | Watts et al. | Oct 2014 | A1 |
| 20140310830 | Zhang et al. | Oct 2014 | A1 |
| 20140315314 | Dubensky, Jr. et al. | Oct 2014 | A1 |
| 20140328819 | Schellenberger et al. | Nov 2014 | A1 |
| 20140329706 | Gale | Nov 2014 | A1 |
| 20140329750 | Andersen et al. | Nov 2014 | A1 |
| 20140335095 | Schneewind et al. | Nov 2014 | A1 |
| 20140348868 | Donati et al. | Nov 2014 | A1 |
| 20140371136 | Schellenberger et al. | Dec 2014 | A1 |
| 20140377289 | Cowan et al. | Dec 2014 | A1 |
| 20150004155 | Beaumont et al. | Jan 2015 | A1 |
| 20150005233 | DeFrees | Jan 2015 | A1 |
| 20150005481 | Chin et al. | Jan 2015 | A1 |
| 20150010566 | Spits et al. | Jan 2015 | A1 |
| 20150023879 | Meinel et al. | Jan 2015 | A1 |
| 20150023959 | Chhabra et al. | Jan 2015 | A1 |
| 20150030594 | Yuan et al. | Jan 2015 | A1 |
| 20150031134 | Zhang et al. | Jan 2015 | A1 |
| 20150031563 | Huynh et al. | Jan 2015 | A1 |
| 20150031604 | Zecri et al. | Jan 2015 | A1 |
| 20150037359 | Schellenberger et al. | Feb 2015 | A1 |
| 20150037421 | Lu et al. | Feb 2015 | A1 |
| 20150037828 | Dulay et al. | Feb 2015 | A1 |
| 20150038421 | Schellenberger et al. | Feb 2015 | A1 |
| 20150045535 | Berka et al. | Feb 2015 | A1 |
| 20150050717 | Collins et al. | Feb 2015 | A1 |
| 20150051082 | Barker et al. | Feb 2015 | A1 |
| 20150056239 | Flechtner et al. | Feb 2015 | A1 |
| 20150056240 | Schneewind et al. | Feb 2015 | A1 |
| 20150071957 | Kelly et al. | Mar 2015 | A1 |
| 20150079132 | Maisonneuve et al. | Mar 2015 | A1 |
| 20150079681 | Zhang | Mar 2015 | A1 |
| 20150086576 | Ploegh et al. | Mar 2015 | A1 |
| 20150087545 | Nair et al. | Mar 2015 | A1 |
| 20150093406 | Del Campo Ascarateil et al. | Apr 2015 | A1 |
| 20150093413 | Thess et al. | Apr 2015 | A1 |
| 20150104468 | Geierstanger et al. | Apr 2015 | A1 |
| 20150118183 | Baumhof | Apr 2015 | A1 |
| 20150118264 | Baumhof et al. | Apr 2015 | A1 |
| 20150132324 | Cong et al. | May 2015 | A1 |
| 20150132335 | Malouin et al. | May 2015 | A1 |
| 20150132339 | Bufali et al. | May 2015 | A1 |
| 20150139984 | Brezski et al. | May 2015 | A1 |
| 20150152134 | Pentelute et al. | Jun 2015 | A1 |
| 20150158929 | Schellenberger et al. | Jun 2015 | A1 |
| 20150165062 | Liao et al. | Jun 2015 | A1 |
| 20150166640 | Lydon | Jun 2015 | A1 |
| 20150168405 | Kojic et al. | Jun 2015 | A1 |
| 20150174130 | Skaar et al. | Jun 2015 | A1 |
| 20150182588 | Kahvejian et al. | Jul 2015 | A1 |
| 20150184142 | Hong et al. | Jul 2015 | A1 |
| 20150185216 | Albert et al. | Jul 2015 | A1 |
| 20150197538 | Janetka et al. | Jul 2015 | A1 |
| 20150197734 | Ma et al. | Jul 2015 | A1 |
| 20150203834 | Iverson et al. | Jul 2015 | A1 |
| 20150210756 | Torres et al. | Jul 2015 | A1 |
| 20150216960 | Zlotnick et al. | Aug 2015 | A1 |
| 20150231228 | Amara et al. | Aug 2015 | A1 |
| 20150232518 | Foster et al. | Aug 2015 | A1 |
| 20150232541 | Fenn et al. | Aug 2015 | A1 |
| 20150232560 | Heindl et al. | Aug 2015 | A1 |
| 20150232561 | Fenn et al. | Aug 2015 | A1 |
| 20150241440 | Fasan et al. | Aug 2015 | A1 |
| 20150246024 | Richter et al. | Sep 2015 | A1 |
| 20150253335 | Burkart et al. | Sep 2015 | A1 |
| 20150258210 | Van Delft et al. | Sep 2015 | A1 |
| 20150259389 | Berka et al. | Sep 2015 | A9 |
| 20150259397 | Lee et al. | Sep 2015 | A1 |
| 20150259431 | Stemmer et al. | Sep 2015 | A1 |
| 20150266943 | Chhabra et al. | Sep 2015 | A1 |
| 20150273040 | McAdow et al. | Oct 2015 | A1 |
| 20150273042 | Maione et al. | Oct 2015 | A1 |
| 20150274800 | Schellenberger et al. | Oct 2015 | A1 |
| 20150284452 | Bremel et al. | Oct 2015 | A1 |
| 20150284477 | Chaikof et al. | Oct 2015 | A1 |
| 20150290362 | Douglas et al. | Oct 2015 | A1 |
| 20150291704 | Beck et al. | Oct 2015 | A1 |
| 20150305361 | Holz-Schietinger et al. | Oct 2015 | A1 |
| 20150306212 | Kahvejian et al. | Oct 2015 | A1 |
| 20150306218 | Nowinski et al. | Oct 2015 | A1 |
| 20150309021 | Birnbaum et al. | Oct 2015 | A1 |
| 20150315248 | Galeotti et al. | Nov 2015 | A1 |
| 20150320882 | Van Delft et al. | Nov 2015 | A1 |
| 20150329568 | Tuttle et al. | Nov 2015 | A1 |
| 20150329590 | Pentelute et al. | Nov 2015 | A1 |
| 20150335724 | Zlotnick et al. | Nov 2015 | A1 |
| 20150338579 | Fattinger | Nov 2015 | A1 |
| 20150343051 | Grandi et al. | Dec 2015 | A1 |
| 20150344862 | Schellenberger et al. | Dec 2015 | A1 |
| 20150346195 | Belmant et al. | Dec 2015 | A1 |
| 20150368322 | McAdow et al. | Dec 2015 | A1 |
| 20150374811 | Malley et al. | Dec 2015 | A1 |
| 20150376266 | Beaumont et al. | Dec 2015 | A1 |
| 20160000895 | Barta et al. | Jan 2016 | A1 |
| 20160002338 | Bossenmaier et al. | Jan 2016 | A1 |
| 20160002346 | Bossenmaier et al. | Jan 2016 | A1 |
| 20160002645 | Clubb et al. | Jan 2016 | A1 |
| 20160018397 | Fischetti et al. | Jan 2016 | A1 |
| 20160022776 | Lee | Jan 2016 | A1 |
| 20160022833 | Bregeon | Jan 2016 | A1 |
| 20160025740 | Song et al. | Jan 2016 | A1 |
| 20160032346 | Tsourkas et al. | Feb 2016 | A1 |
| 20160038581 | Bielke et al. | Feb 2016 | A1 |
| 20160040158 | Wagner et al. | Feb 2016 | A1 |
| 20160041157 | Tsourkas et al. | Feb 2016 | A1 |
| 20160045885 | Love et al. | Feb 2016 | A1 |
| 20160052982 | Cohen et al. | Feb 2016 | A1 |
| 20160068583 | Van Vlasselaer et al. | Mar 2016 | A1 |
| 20160068589 | Lydon | Mar 2016 | A1 |
| 20160068591 | Anderson et al. | Mar 2016 | A1 |
| 20160069894 | Smider et al. | Mar 2016 | A1 |
| 20160073671 | Geistlinger et al. | Mar 2016 | A1 |
| 20160074497 | Falugi et al. | Mar 2016 | A1 |
| 20160074498 | Hultgren et al. | Mar 2016 | A1 |
| 20160082046 | Lodish et al. | Mar 2016 | A1 |
| 20160090351 | Hedstrom et al. | Mar 2016 | A1 |
| 20160090404 | Malley et al. | Mar 2016 | A1 |
| 20160097773 | Pasqual et al. | Apr 2016 | A1 |
| 20160102137 | Bjorkman et al. | Apr 2016 | A1 |
| 20160102332 | Collier et al. | Apr 2016 | A1 |
| 20160102344 | Niemeyer et al. | Apr 2016 | A1 |
| 20160108091 | Zecri et al. | Apr 2016 | A1 |
| 20160114046 | Brudno et al. | Apr 2016 | A1 |
| 20160115222 | Lydon | Apr 2016 | A1 |
| 20160115488 | Zhang et al. | Apr 2016 | A1 |
| 20160115489 | Zhang et al. | Apr 2016 | A1 |
| 20160122405 | Palchaudhuri et al. | May 2016 | A1 |
| 20160122451 | Chilkoti et al. | May 2016 | A1 |
| 20160122707 | Swee et al. | May 2016 | A1 |
| 20160123991 | Mumm et al. | May 2016 | A1 |
| 20160129101 | Biemans et al. | May 2016 | A1 |
| 20160130299 | Perez et al. | May 2016 | A1 |
| 20160136298 | Grawunder et al. | May 2016 | A1 |
| 20160137698 | Skerra et al. | May 2016 | A1 |
| 20160137711 | Schellenberger et al. | May 2016 | A1 |
| 20160137720 | Song et al. | May 2016 | A1 |
| 20160146786 | Hopkins et al. | May 2016 | A1 |
| 20160146794 | Johnsson et al. | May 2016 | A1 |
| 20160158335 | Bagnoli et al. | Jun 2016 | A1 |
| 20160166634 | Caplan et al. | Jun 2016 | A1 |
| 20160168232 | Beaumont et al. | Jun 2016 | A9 |
| 20160175412 | Von Boehmer et al. | Jun 2016 | A1 |
| 20160175441 | Schneewind et al. | Jun 2016 | A1 |
| 20160178627 | Albert et al. | Jun 2016 | A1 |
| 20160184421 | Huang et al. | Jun 2016 | A1 |
| 20160185791 | Nicolaou et al. | Jun 2016 | A1 |
| 20160185817 | Zhu et al. | Jun 2016 | A1 |
| 20160185828 | Joshi et al. | Jun 2016 | A1 |
| 20160193355 | Qin et al. | Jul 2016 | A1 |
| 20160194363 | Schneewind et al. | Jul 2016 | A1 |
| 20160194410 | Gallusser et al. | Jul 2016 | A1 |
| 20160194627 | Smider et al. | Jul 2016 | A1 |
| 20160199454 | Liu et al. | Jul 2016 | A1 |
| 20160199510 | McDonald et al. | Jul 2016 | A1 |
| 20160200742 | Zhang et al. | Jul 2016 | A1 |
| 20160206566 | Lu et al. | Jul 2016 | A1 |
| 20160208233 | Liu et al. | Jul 2016 | A1 |
| 20160213744 | Liu et al. | Jul 2016 | A1 |
| 20160220686 | Brudno et al. | Aug 2016 | A1 |
| 20160222096 | Beaumont et al. | Aug 2016 | A1 |
| 20160230216 | Nair et al. | Aug 2016 | A1 |
| 20160244747 | Liu et al. | Aug 2016 | A1 |
| 20160244784 | Jacobson et al. | Aug 2016 | A1 |
| 20160251409 | Oestergaard et al. | Sep 2016 | A1 |
| 20160257749 | Lifke et al. | Sep 2016 | A1 |
| 20160257932 | Kahvejian et al. | Sep 2016 | A1 |
| 20160264624 | Cong et al. | Sep 2016 | A1 |
| 20160271268 | Shih et al. | Sep 2016 | A1 |
| 20160279192 | Saito et al. | Sep 2016 | A1 |
| 20160279257 | Koussa et al. | Sep 2016 | A1 |
| 20160280748 | Liu et al. | Sep 2016 | A1 |
| 20160282369 | Cravatt et al. | Sep 2016 | A1 |
| 20160287734 | Rashidian et al. | Oct 2016 | A1 |
| 20160297854 | Ghosh et al. | Oct 2016 | A1 |
| Entry |
|---|
| Chatlin et al., Delivery of antisense oligonucleotides using cholesterol-modified sense dendrimers and cationic lipids, Bioconjugate Chem 16, 827-836. (Year: 2005). |
| Burglin, T., The hedgehog protein family, Genome Biology, 9:241 (doi:10.1186/GB-2008-9-11-241) (Year: 2008). |
| Number | Date | Country | |
|---|---|---|---|
| 20200377920 A1 | Dec 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62409655 | Oct 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15782391 | Oct 2017 | US |
| Child | 16989843 | US |
