Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0056—Peptides, proteins, polyamino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/104—Aminoacyltransferases (2.3.2)
  • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/02—Aminoacyltransferases (2.3.2)
  • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

• Transglutaminase 2 (TG2) is a member of the human transglutaminase family of enzymes, which is abundantly expressed in various tissues and is found in both intra- and extra- cellular locations (Lorand and Graham, 2003). It possesses the catalytic activity of deamidating glutamine sidechains on substrate peptides or proteins or crosslinking them with biogenic small molecule or protein-bound amines.
• TG2 substrate is SEQ ID NO:1, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, a 33-mer gluten peptide that reveals HLA- DQ2 epitopes upon TG2-catalyzed deamidation at the underlined glutamine residues (Shan et al., 2002).
• TG2 has been attributed with various biological functions (for reviews, see e.g. Iismaa et al., 2009; Nurminskaya & Belkin, 2012).
• compositions and methods for in vivo or ex vivo delivery of a molecular cargo into endo-lysosomal compartments of LRP1-expressing cells are provided herein.
• the molecular cargo is linked to a substrate or inhibitor for recognition and transport via human transglutaminase 2 (TG2).
• TG2 human transglutaminase 2
• the compositions of the disclosure can be incorporated into formulations for therapeutic and diagnostic applications.
• the compositions also provide a flexible platform for delivery of a cargo of interest to lysosomal compartments.
• the disclosure herein demonstrates a novel biological mechanism by which a TG2 substrate or inhibitor can undergo receptor-mediated endocytosis in a TG2-dependent manner.
• TG2 binds to gluten and gluten-like molecules in the extracellular matrix.
• the enzyme- substrate/inhibitor complex is recognized by the plasma protein ⁇ 2-macroglobulin, and the resulting ternary complex is endocytosed via recognition by the cluster I/II of LRP1 (low-density lipoprotein receptor-related protein-1).
• LRP1 low-density lipoprotein receptor-related protein-1
• the TG2 substrate/inhibitor and linked cargo can be delivered to lysosomes, and may be presented on the cell-surface in an MHC-restricted context.
• the methods and compositions disclosed herein utilize this pathway to provide for enhanced endo-lysosomal uptake by cells expressing LRP1 of molecular cargo linked to a TG2 substrate or inhibitor.
• the TG2 substrate/inhibitor comprises or consists of a polypeptide comprising the pentapeptide sequence Pro-X-Y-Pro-R (formula I), where X is selected from amino acids that engage TG2 through the formation of a covalent enzyme-compound intermediate, including without limitation glutamine, ⁇ -diazoketones, ⁇ -halo- ketones, ⁇ -unsaturated carbonyl compounds, and ⁇ -unsaturated sulfones (see formula II, amino acid X).
• Y is any amino acid, including unnatural amino acids with easily appended functional groups, including without limitation primary/secondary amines, alcohols, and carboxylic acids;
• R is a natural or non-natural aromatic amino acid, e.g. tyrosine (Y), phenylalanine (F), and tryptophan (W), naphthalene, etc.;
• the C-terminus of the pentapeptide can be a carboxylic acid, an ester, or an amide; wherein molecular cargo is linked to the C-terminus of the pentapeptide, and/or to Y.
• the cargo may be joined to the pentapeptide through a linker, through covalent direct conjugation, through non-covalent high affinity pairing, and the like.
• Molecular cargo include any compound that is of interest for delivery to a cell.
• Cargo moieties of interest include, without limitation, antibiotics, antiviral agents, chemotherapeutic agents, nucleosides, polynucleotides, e.g.
• siRNA, mRNA, DNA, etc. proteins, fluorescent/radioactive/optical imaging agents, peptides/proteins/enzymes, nucleic acids (siRNA/RNA/DNA/etc.), metal based compounds/catalysts, polymers, site-specific cellular targeting agents (compounds/ligands/antibodies/etc.), immunomodulatory drugs, antigen binding moieties, antibody binding moieties, etc., and are useful for diverse applications such as imaging, treating infection, chemotherapeutic agents, smart adjuvants, gene therapy vectors, biosensors, bioreactors, and so forth.
• Molecular cargo can be joined to the substrate/inhibitor through a linker, optionally a cleavable linker.
• a TG2 substrate/inhibitor as disclosed herein comprising linker as molecular cargo, where the linker can be activated to join the substrate/inhibitor to an agent of interest.
• the TG2 substrate/inhibitor comprises or consists of Formula II: where X is selected from: where R 1 is selected from Cl, Br, I, OSO 2 CF 3 ; R 2 is selected from N(CH 3 ) 2 , OCH 3 , NH 2 , CH 3 , etc.; Y is any amino acid, R is an aryl, heteroaryl or substituted aryl group.
• R is Ph, CH 2 Ph (Phe, F), CH2PhOH (Tyr, Y), CH2Naphthelene; and Z is OH, OR 3 , NH2, NHR 3 , N(R 3 )2, etc., or Z is molecular cargo or a linker.
• R 3 is an alkyl, e.g. a lower alkyl, substituted alkyl, heteroalkyl, etc.
• the compounds disclosed herein, i.e. a TG2 substrate or inhibitor optionally joined to a molecular cargo directly or through a suitable linker can be provided as a formulation in combination with a pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants.
• Formulations may be provided for oral, parenteral, etc. administration. Formulations may be provided in a unit dosage, e.g. in an effective dose for imaging, therapy, and the like.
• compositions and methods are provided for imaging TG2 in vitro or in vivo, the method comprising contacting a subject, tissues, cells, biological fluids, etc. suspected of comprising catalytically active TG2 with an effective dose of a TG2 substrate or inhibitor joined to molecular cargo for imaging, where imaging moieties include without limitation radiography moieties, e.g.
• the imaging moiety is then detected in the tissues, cells, etc. In some embodiments the imaging is performed in vivo.
• the TG2 substrate or inhibitor and cargo allow for direct visualization of catalytically active TG2 in vivo.
• the electrophilic amino acid X is selected to comprise an isosteric unnatural amino acid harboring an ⁇ , ⁇ -unsaturated dimethylamide “soft” electrophile. The resulting non-hydrolyzable adduct thus labels TG2 directly for analysis.
• compositions and methods are provided for the delivery of molecular cargo into endo-lysosomal compartments of LRP1-expressing cells, the method comprising contacting a subject, tissues, cells, biological fluids, etc.
• the IgG binding moiety can be pre- complexed with a compound of interest linked to an IgG, for delivery of the compound of interest to the endo-lysosomal compartment.
• Such methods can provide for treatment, for example, of disorders of endolysosomal metabolism such as lysosomal storage diseases.
• FIG.12A-12C (A) Treating NRK cells with increasing concentrations of HB-3-003 in the presence of fluorescently tagged streptavidin leads to increased internalization of streptavidin after a 90min treatment. (B) Quantification of dose response. (C) Biotin and PQLPF alone do not lead to increased uptake of streptavidin. [0026]
• FIG.12A-12C (A) Treating NRK cells with increasing concentrations of HB-3-002 leads to decrease of cell membrane cubilin after a 90min treatment. (B) Quantification of dose response. (C) Vitamin B 12 and PQLPF alone do not lead to increased uptake of cubilin. [0027] FIG.
• FIGS.14A-14B Metabolic stability of HB-230 and HB-2121.
• A HB-230 is transformed by chymotrypsin to give the C-terminal carboxylic acid. This metabolite is subsequently cleaved by carboxypeptidase to give the tetrapeptide without the terminal phenylalanine.
• B HB-2121 is stable to both chymotrypsin and carboxypeptidase mediated metabolism.
• NRK cells were treated with 2 ⁇ M HB-2121 (red) with or without 100 ⁇ g/mL ⁇ -2-macrogloblulin, showing ⁇ 2M-enhanced endocytic uptake of the TG2 probe, as was seen with HB-230.
• DETAILED DESCRIPTION OF THE EMBODIMENTS [0030] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
• a cell includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
• the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. None herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
• the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like.
• "Mammal” means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans.
• Non-human animal models e.g., mammals, e.g.
• prediction is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.
• treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease.
• Treating may include treatment of cancer in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms.
• Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
• the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
• treating includes the administration of engineered cells to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with disease or other diseases.
• therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
• a "therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder.
• a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease.
• a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
• a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
• the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
• a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
• the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.
• the invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.
• the immunoglobulin single variable domain includes fully human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences.
• Such immunoglobulin single variable domains may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V HH sequences (i.e., from a suitable species of Camelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g., from human).
• V HH sequences i.e., from a suitable species of Camelid, e.g., llama
• VHs or VLs e.g., from human
• the TG2 substrate/inhibitor and molecular cargo may be separated by a linker, e.g. a polypeptide linker, or a non-peptidic linker, etc.
• the linker is a rigid linker, in other embodiments the linker is a flexible linker.
• the linker moiety is a peptide linker.
• a peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids.
• a “cleavable linker” is a linker that has one or more cleavable groups that may be broken by the result of a reaction or condition.
• the term “cleavable group” refers to a moiety that allows for release of a component of the solid support or oligomer of the invention by cleaving a bond linking the released moiety to the remainder of the conjugate. Exemplary cleavage mechanisms of use both in preparing and using the oligomers and solid supports of the invention are enzymatically or otherwise chemically mediated.
• non-enzymatic cleavage agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamate esters, benzoin esters), and heat.
• light e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamate esters, benzoin esters
• heat e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamate esters, benzoin esters
• Many cleaveable groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., E
• cleavable, bifunctional (both homo- and hetero-bifunctional) spacer arms are commercially available.
• An exemplary cleavable group is cleavable by a reagent, e.g. sodium hydroxide, ammonia or other amine.
• the cleavable linker is readily cleaved at room temperature or under heat.
• linker is a valine-citrulline dipeptide linker that is cleaved by cathepsin B in the lysosome.
• Chemical groups that find use in linking binding domains include carbamate; amide (amine plus carboxylic acid); ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like, as known in the art.
• the linkage between binding domains may comprise spacers, e.g. alkyl spacers, which may be linear or branched, usually linear, and may include one or more unsaturated bonds; usually having from one to about 300 carbon atoms; more usually from about one to 25 carbon atoms; and may be from about three to 12 carbon atoms.
• Spacers of this type may also comprise heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like.
• linkers may include polyethylene glycol, which may be linear or branched.
• the TG2 substrate/inhibitor may comprise a homo- or heterobifunctional linker having a group at one end capable of forming a stable linkage to cargo.
• Illustrative entities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis- sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- ⁇ - maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N- succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde
• succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue.
• Other reagents useful for this purpose include: p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4-disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); disdiazobenzidine (which reacts primarily with tyrosine and histidine); O-benzotriazolyloxy tetramethuluronium hexafluorophosphate (HATU
• Homobifunctional cross-linking reagents include bismaleimidohexane ("BMH").
• “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, naturally occurring or non-naturally occurring, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa.
• a small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.
• polypeptide and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
• nucleic acid molecule refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
• Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
• a “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder.
• the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
• a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
• “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
• a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
• a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
• Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
• the term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells.
• the definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.
• biological sample encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.
• a “biological sample” includes a sample obtained from a patient’s diseased cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising diseased cells from a patient.
• a biological sample comprising a diseased cell from a patient can also include non-diseased cells.
• each component When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
• Conscomitant administration means administration of one or more components, such as engineered proteins and cells, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.
• a first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.
• a TG2 substrate/inhibitor of the disclosure comprises or consists of a polypeptide comprising the pentapeptide sequence Pro-X-Y-Pro-R (formula I), where X is selected from amino acids that engage TG2 through the formation of a covalent enzyme-compound intermediate, including without limitation glutamine, ⁇ -diazoketones, ⁇ -halo-ketones, ⁇ - unsaturated carbonyl compounds, and ab-unsaturated sulfones.
• X is glutamine.
• Y is any amino acid, including unnatural amino acids with easily appended functional groups, including without limitation primary/secondary amines, alcohols, and carboxylic acids).
• R is a natural or non-natural aromatic amino acid, e.g. tyrosine (Y), phenylalanine (F), and tryptophan (W), naphthalene, etc.
• the C-terminus of the pentapeptide can be a carboxylate, an ester, or an amide.
• Molecular cargo or a linker is linked to the C-terminus of the pentapeptide, and/or to Y.
• the cargo may be joined to the pentapeptide through a linker, through covalent direct conjugation, through non-covalent high affinity pairing, and the like.
• a TG2 substrate/inhibitor comprises a linker suitable for joining to cargo.
• Molecular cargo include any compound that is of interest for delivery to a cell.
• Cargo moieties of interest include, without limitation, antibiotics, antiviral agents, chemotherapeutic agents, nucleosides, polynucleotides, e.g.
• siRNA, mRNA, DNA, etc. proteins, fluorescent/radioactive/optical imaging agents, peptides/proteins/enzymes, nucleic acids (siRNA/RNA/DNA/etc.), metal based compounds/catalysts, polymers, site-specific cellular targeting agents (compounds/ligands/antibodies/etc.), immunomodulatory drugs, antigen binding moieties, etc., and are useful for diverse applications such as imaging, treating infection, chemotherapeutic agents, smart adjuvants, gene therapy vectors, biosensors, bioreactors, and so forth.
• Molecular cargo can be joined to the substrate/inhibitor through a linker, optionally a cleavable linker.
• the TG2 substrate/inhibitor comprises or consists of Formula II: where X is selected from: where R 1 is selected from Cl, Br, I, OSO 2 CF 3 ; R 2 is selected from N(CH 3 ) 2 , OCH 3 , NH 2 , CH 3 , etc.; Y is any amino acid, R is an aryl, heteroaryl or substituted aryl group. In some embodiments R is Ph, CH 2 Ph, CH2PhOH, CH2Naphthelene. and Z is OH, OR 3 , NH2, NHR 3 , etc., or Z is molecular cargo or a linker. R 3 is an alkyl, e.g.
• an imaging composition comprising or consisting of a TG2 substrate/inhibitor of the disclosure, linked to an imaging moiety.
• Imaging moieties are as described herein, and include without limitation radiography moieties, e.g. heavy metals and radiation emitting moieties, positron emitting moieties, magnetic resonance contrast moieties, and optically visible moieties, e.g., fluorescent or visible-spectrum dyes, visible particles, etc.
• an imaging composition is AcPro-HWE-(Dab-SCy5)-Pro-Phe (“HB-230”), where HWE is the unnatural amino acid (S,E)-2-amino-7-(dimethylamino)-7-oxohept-5-enoic acid, Dab is the unnatural amino acid (S)-2,4-diaminobutanoic acid, and SCy5 is the small molecule fluorophore sulfo-cyanine 5.
• a compound of interest is HB-230 or HB-2121, as shown below.
• a targeting composition comprising a TG2 substrate/inhibitor of the disclosure, linked to a targeting moiety, e.g. a binding domain specific for a targeted molecule.
• a targeting moiety e.g. a binding domain specific for a targeted molecule.
• Specific binding domains are as described herein, and may comprise, for example, an antibody or fragment thereof that specifically binds to a targeted polypeptide.
• a targeted polypeptide is a pathogenically relevant extracellular protein.
• a compound of interest specifically targeted to IgG proteins is HB-328:
• a therapeutic composition is provided, comprising a TG2 substrate/inhibitor of the disclosure, linked to therapeutic moiety, e.g.
• molecular cargo comprising a small molecule drug, such as chemotherapeutic, antibiotic, anti-viral, etc.
• the cargo may be joined to the TG2 substrate/inhibitor through a linker.
• the linker may be a linker that is cleaved in the lysosomal complex, thereby releasing the cargo.
• the substrate or inhibitor, optionally with a linker or cargo may be provided in a pharmaceutical composition.
• the pharmaceutical composition may be provided in a unit dose. Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters.
• the dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.
• the TG2 substrate or inhibitor and cargo can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal.
• Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.
• An agent can be administered in any manner which is medically acceptable.
• a TG2 substrate or inhibitor and cargo can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application).
• a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
• An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
• a TG2 substrate or inhibitor and cargo can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application.
• compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
• diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
• the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
• compounds which are "commercially available” may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc.
• the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
• the agents may be combined, as previously described, to provide a cocktail of activities.
• the following methods and excipients are exemplary and are not to be construed as limiting the invention.
• the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
• conventional additives such as lactose, mannitol, corn starch or potato starch
• binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
• disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
• lubricants such as talc or magnesium stearate
• the oral formulations comprise enteric coatings, so that the active agent is delivered to the intestinal tract.
• enteric coatings A number of methods are available in the art for the efficient delivery of enterically coated proteins into the small intestinal lumen. Most methods rely upon protein release as a result of the sudden rise of pH when food is released from the stomach into the duodenum, or upon the action of pancreatic proteases that are secreted into the duodenum when food enters the small intestine.
• the enzyme is usually lyophilized in the presence of appropriate buffers (e.g. phosphate, histidine, imidazole) and excipients (e.g.
• cryoprotectants such as sucrose, lactose, trehalose. Lyophilized enzyme cakes are blended with excipients, then filled into capsules, which are enterically coated with a polymeric coating that protects the protein from the acidic environment of the stomach, as well as from the action of pepsin in the stomach. Alternatively, protein microparticles can also be coated with a protective layer. Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate, methacrylate copolymers, and cellulose acetate phthalate.
• enteric formulations comprise engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings and can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches.
• the polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623): 410- 414.
• Drug delivery systems can also utilize a core of superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh et al.
• Formulations are typically provided in a unit dosage form, where the term "unit dosage form,” refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
• the specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
• the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are commercially available.
• auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.
• Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt.
• “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
• Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
• Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
• compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
• a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
• Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties.
• the nature of the carrier can be either soluble or insoluble for purposes of the invention.
• methods are provided for linking a molecular cargo of interest to a TG2 substrate/inhibitor, for delivery of the molecules to endo-lysosomal compartments of LRP1- expressing cells.
• a TG2 substrate/inhibitor is provided, comprising a linker suitable for attaching a molecular cargo of interest.
• a TG2 substrate/inhibitor is provided comprising an affinity reagent, e.g. biotin, for attaching avidin- conjugated cargo.
• NRK cells were co- incubated with fluorescent ⁇ 2 M and the aforementioned 33-residue gluten peptide (33mer) tagged with a Sulfo- Cy5 fluorophore.
• 33mer 33-residue gluten peptide
• LRP-1 Inhibiting LRP-1 through RAP mitigates the depletion of surface cubilin. Individual components alone (vitamin B 12 and PQLPF) do not promote the depletion of cubilin.
• Targeted intracellular delivery of a payload through the LRP-1 pathway We synthesized HB2108, by linking AcPQLPF to Cy5 via a cathepsin-labile valine-citrulline linker. NRK cells were t hen treated with HB2108 and ⁇ 2M in the presence and absence of a cathepsin B inhibitor.
• Example 2 [00159] Extensive work has intimately linked the enzymatic activity of transglutaminase 2 (TG2) to the pathogenesis of celiac disease (CeD). TG2 performs the site-selective deamidation of glutamine side chains (Gln ⁇ Glu) within wheat-derived peptides, rendering them more immunogenic in the genetic background of CeD. Additionally, recent work has shown that the amount of catalytically active TG2 in the small intestine of an individual may correlate to CeD severity. These features of TG2 have inspired the initiation of multiple drug discovery campaigns to develop novel agents to inhibit the enzyme in hopes of unlocking a novel therapeutic modality for CeD.
• TG2 transglutaminase 2
• CeD celiac disease
• TG2 can mediate the endolysosomal delivery of gluten antigens in the context of CeD pathogenesis.
• HB-2121 is analogous to the TG2 inhibitor/probe HB-230 but with one modification; the primary amide C-terminus of HB-230 was replaced with a tertiary dimethyl amide. This change greatly enhances the gastrointestinal stability of the probe.
• HB-230 suffered from enzymatic deamidation at the C-terminus (CONH 2 ⁇ CO 2 H) by chymotrypsin and subsequent cleavage of the C-terminal phenylalanine by carboxypeptidase. Modification of the C-terminus of HB-230 to give HB-2121 allowed complete resistance to metabolism by chymotrypsin and thus no subsequent metabolism by carboxypeptidase, shown in FIG.14.
• TG2-inhibitor complexes are potently endocytosed in an ⁇ -2- macrogloblulin-dependent manner by the LRP-1 receptor.
• This endocytic mechanism represents a novel avenue by which cargoes of interest can be shuttled into the lysosome for delivery or degradation.
• a bifunctional small molecule HB328
• HB328 bifunctional small molecule that on one end engages TG2 through covalent inhibition and on the other non-covalently binds the Fc region of IgG antibodies.
• the TG2 engaging half features the same pentapeptide scaffold as in HB-230.
• the Fc binder consists of a doubly disulfide linked 15 amino acid bicyclic peptide that has been previously characterized by Gong et al. (2016) Bioconjugate Chem.2016, 27 (7), 1569– 1573.
• affinity KD 3 nM.
• Structure of HB328 [00163] The ability of this compound to bring about endocytosis of any IgG gives it significant modularity in both a research and therapeutic context.
• One such therapeutic use is in the removal of maladaptive IgG that occurs in the context of rheumatologic disease (eg. rheumatoid arthritis, idiopathic thrombocytopenic purpura, etc.).
• HB-328 can allow the lysosomal delivery of recombinant proteins by co-dosing HB-328 with the protein of interest, linked to an IgG Fc domain. This allows treatment of disorders of endolysosomal metabolism such as the lysosomal storage diseases.

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