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
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • 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
  • A61K47/641—Branched, dendritic or hypercomb peptides
• • 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
  • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• • 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
  • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

• Saporin is a ribosome inactivating protein (RIP, 34 KD) with no cell entry domain. Given an entry mechanism into the cell, it catalytically removes a single adenine from ribosomal RNA of the large subunit of the ribosome, completely inactivating it. As such, if it can be delivered inside the cell, it is a potent toxin that rapidly kills the cell.
• RIP ribosome inactivating protein
• Described herein are targeted cancer therapies comprising one or more MGS peptides and the plant toxin saporin.
• the compositions described herein can, in some aspects, bind to and mediate internalization of the compositions into cancer cells resulting in rapid cell death. Because of the cancer specificity of the MGS peptides, the treatment has minimal effect on normal tissues.
• compositions described herein can be created as a fusion protein or by chemical conjugation.
• the conjugate can be delivered and the MGS directs the saporin to the tumor and mediates its uptake in the tumor cells where it can bind its target. This compound has several advantages over current therapies.
• the disclosed compositions (1) can allow for a highly potent cytotoxic to be delivered intracellularly where it is functional; (2) the MGS can be cancer specific minimizing uptake of the toxin in other tissues; (3) because all cells, even non dividing cells, are dependent on protein synthesis, the MGS peptide-saporin conjugate can be effective against cells that are refractory to common cytotoxic agents; (4) resistance is unlikely due to the mechanism of action as a protein, multiple drug resistance pumps are unlikely to negate activity; and (5) the peptidic targeting agent is small, relatively inexpensive compare to antibody targeting agents, and can be conjugated to saporin.
• compositions comprising one or more a molecular guidance system (MGS) peptides and a cytotoxic agent.
• MMS molecular guidance system
• membrane-permeable conjugates for transport across a lipid membrane comprising: one or more a molecular guidance system (MGS) peptides and a cytotoxic agent.
• MMS molecular guidance system
• cytotoxic agent targets an intracellular target.
• FIG. 1 shows that HCC15.2 is specific for and internalizes in cancer cells.
• FIG. 2 shows that HCC15.2 binds to all subtypes of NSCLC.
• FIG. 3 shows that peptide internalization is receptor mediated.
• FIG. 4 shows that tetramerization of SEQ ID NO: 1 does not significantly improve internalization compared to the monomer of SEQ ID NO: 1.
• FIG. 5 shows that truncations from N- and C-Terminal ends reveal minimal binding sequence (FHAVPQSFYTAP, SEQ ID NO: 1; FHAVPQSFYTA, SEQ ID NO: 2; and FHAVPQSFYT, SEQ ID NO: 3).
• FIG. 6 shows that acetylation improves binding of the truncated peptide (SEQ ID NO: 3, FHAVPQSFYTAP, SEQ ID NO: 1).
• FIG. 7 shows that peptide co-localizes with the lysosome.
• FIG. 8 shows that HCC15.2 accumulates in the lysosome over time.
• FIG. 9 shows that in vitro drug data of HCC15.2 conjugated to saporin.
• FIG. 10 shows that the HCC15.2-saporin conjugate reduces tumor growth in a human NSCLC xenograft model.
• FIG. 11 depicts a histogram and a quantification of a histogram from flow cytometry experiments.
• FIG. 12 is a bar graph showing that HCC15.2 binds to other cancer cell lines.
• FIG. 13 is a line graph showing the flow cytometry results to measure KD.
• FIG. 14 shows that tetramerization does not significantly improve half maximal binding.
• FIG. 15 shows that truncations from N- and C-Termini reveal minimal binding sequence.
• Top panel refers to the following sequences from top to bottom: SEQ ID NOs: 1, 2, 3, 78, and 79.
• Bottom panel refers to the following sequences from top to bottom: SEQ ID NOs: 1, 2, 3, 78, 79, and 80.
• FIG. 16 shows that HCC15.2 accumulates in the lysosome over time.
• FIG. 17 shows that HCC15.2-targeted NIR dye accumulates into tumors in vivo.
• FIG. 18 shows that HCC 15.2-targerted dye is retained in the tumor over time.
• FIG. 19 demonstrates that ex vivo tumors show clear accumulation in HCC 15.2- targeted dye.
• FIG. 20 shows the results of ex vivo imaging of organs.
• FIG. 21 shows that HCC 15.2 targets saporin to cancer cells in vitro.
• FIG. 22 is an example of the cancer cell killing assay showing that HCC15.2 targets saporin to cancer cells in vitro.
• FIG. 23 shows that HCC 15.2 targeting of saporin markedly slowed tumor growth.
• FIG. 24 is a table listing various MGSs, indication, cell type targets, cellular location and payloads delivered.
• FIG. 25 shows sequences (in order from top to bottom: SEQ ID NOs: 22, 39, 40, 23, 47, 25, 26, 27, 28, and 29) cellular location, valency, cancer specificity and binding profile information for select MGS peptides.
• FIG. 26 is a table showing further characterization of select MGS peptides.
• FIG. 27 shows co-localization of H1299.3 15-mer with autophagosomes (top panel) and results after administration in vivo.
• FIG. 28 is a bar graph showing 1299.3 Ac-15mer cancer cell binding compared to normal cell binding.
• FIG. 29 is a table showing saporin delivery in vitro with other peptides (parental peptide not optimized except for HCC15.2).
• FIG. 30 is an example of a multimeric MGS peptide and experimental results using the multimeric MGS peptide (SEQ ID NO: 22).
• MGS peptide means any one member of a particular list and also includes any combination of members of that list.
• reference to “a MGS peptide” includes a plurality of such MGS peptides
• reference to “the MGS peptide” is a reference to one or more MGS peptides and equivalents thereof known to those skilled in the art, and so forth.
• Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to "about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as "about” that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then “about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.
• the term "subject” refers to the target of administration, e.g., a human.
• the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
• the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
• a subject is a mammal.
• a subject is a human.
• the term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
• the term "patient” refers to a subject afflicted with a disease or disorder.
• the term “patient” includes human and veterinary subjects.
• the "patient” has been diagnosed with a need for treatment for an autoimmune disorder, such as, for example, prior to the administering step.
• amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues.
• the amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.
• Polypeptide refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term “polypeptide” encompasses naturally occurring or synthetic molecules.
• polypeptide refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids.
• the polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications.
• Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
• nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing.
• Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodi ester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
• nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
• sample is meant to mean an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein.
• a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
• module is meant to mean to alter, by increasing or decreasing.
• an effective amount of a compound is meant to mean a sufficient amount of the compound to provide the desired effect.
• the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
• isolated polypeptide or “purified polypeptide” is meant to mean a polypeptide (or a fragment thereof) that is substantially free from the materials with which the polypeptide is normally associated in nature.
• the polypeptides of the invention, or fragments thereof can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide.
• polypeptide fragments may be obtained by any of these methods, or by cleaving full length proteins and/or polypeptides.
• isolated nucleic acid or “purified nucleic acid” is meant to mean DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene.
• the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis).
• isolated nucleic acid also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or polypeptide molecules.
• treat is meant to mean administer a compound or molecule of the invention to a subject, such as a human or other mammal (for example, an animal model), that has a cancer, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease.
• prevent is meant to mean minimize the chance that a subject who has an increased susceptibility for developing cancer will develop cancer.
• an antibody recognizes and physically interacts with its cognate antigen or target (for example, the disclosed synthetic MGS sequences) and does not significantly recognize and interact with other antigens or targets; such an antibody may be a polyclonal antibody or a monoclonal antibody, which are generated by techniques that are well known in the art.
• probe As used herein, "probe,” “primer,” or “oligonucleotide” is meant to mean a single- stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target”).
• the stability of the resulting hybrid depends upon the extent of the base-pairing that occurs.
• the extent of base- pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions.
• the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art.
• Probes or primers specific for nucleic acids capable of encoding the disclosed MGS sequences have at least 80%-90% sequence complementarity, preferably at least 9 ⁇ %-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid capable of encoding the disclosed MGS sequences to which they hybridize.
• Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art.
• Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).
• SSCP single stranded conformational polymorphism
• RFLP restriction fragment polymorphism
• Southern hybridization Southern hybridization
• Northern hybridization in situ hybridization
• ESA electrophoretic mobility shift assay
• telomere sequence a nucleic acid capable of encoding the disclosed MGS sequence
• a substantially complementary nucleic acid for example, a nucleic acid capable of encoding the disclosed MGS sequence
• high stringency conditions is meant to mean conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP0 4 , pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65°C, or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C.
• compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning
• any subset or combination of these is also disclosed.
• the subgroup of A-E, B-F, and C-E would be considered disclosed.
• This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
• steps in methods of making and using the disclosed compositions are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
• compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.
• a targeted cancer therapy comprising a peptide conjugate comprising the peptide sequence FHAVPQSFYT (SEQ ID NO: 3) conjugated to the plant toxin saporin.
• Saporin is a ribosome inactivating protein (RIP, 34 KD) with no cell entry domain. Given an entry mechanism into the cell, it catalytically removes a single adenine from ribosomal RNA of the large subunit of the ribosome, inactivating it. As such, if it can be delivered inside the cell, it is a potent toxin that rapidly kills the cell.
• this problem can be overcome by conjugating saporin to a peptide that is selective for cancer cells and binds to and mediates internalization of the toxin into cancer cells, thus resulting in rapid cancer cell death. Because of the cancer specificity of the MGS peptide conjugate disclosed herein, the treatment has minimal effect on normal tissues.
• Disclosed herein is an anti-cancer therapy with applications in lung, breast, colorectal, ovarian, and pancreatic cancers.
• the compound, composition, or peptide conjugate disclosed herein represents a targeted cytotoxic therapy that may have wide applicability in the treatment of carcinomas.
• composition or conjugate e.g., the MGS peptide-saporin conjugate
• the conjugate can be delivered and the MGS peptide can direct the toxin, (e.g., saporin) to the tumor or cancer cell where it mediates the uptake of the saporin into the tumor cells where the toxin can bind its target.
• toxin e.g., saporin
• compositions disclosed herein for example, when one or more molecular guidance system (MGS) peptides when coupled or conjugated to saporin can target and deliver saparoin specifically to cancer cells while avoiding or minimizing the uptake of saporin in normal cells.
• MGS peptides disclosed herein have a low nanomolar affinity for a subset of epithelial-derived cancers. Because these MGS peptides are small, they can be easily conjugated to saporin or expressed as a fusion protein. With about 1.6 million new diagnoses and almost 600,000 deaths per year in the U.S. resulting in annual health care costs of approximately $89 billion, the conjugates disclosed herein provide a new therapy for carcinoma that may overcome the limitations of current cancer therapies.
• the peptide HCC15.2 can be truncated by removing removal of -one or more amino acids. Further a PEG linker can be added to the monomelic peptide, and the amino terminus can be acetylated.
• MGS peptides Disclosed herein are molecular system guidance peptides (MGS) or tumor targeting peptides. These peptides can bind selectively to tumors including malignant tumors.
• MGS peptides that can be used or modified in the disclosed compositions can include, but are not limited to, one or more of the MGS peptides disclosed in McGuire et al, Sci Rep. 2014 Mar 27; 4:4480.
• MGS peptides that can also be used in the disclosed compositions and methods, include, but are not limited to the MGS sequences shown in Table 1 and FIGS. 24 and 25.
• compositions comprise one or more a molecular guidance system (MGS) peptides and a cytotoxic agent.
• MMS molecular guidance system
• the membrane-permeable conjugates for transport across a lipid membrane can comprise one or more a molecular guidance system (MGS) peptides and a cytotoxic agent.
• the one or more MGS peptides can be any of the MGS peptides disclosed herein.
• the one or more MGS peptides comprise SEQ ID NO: 1, 2, 3,
• the one or more MGS peptides comprise SEQ ID NOs: 1, 2 or 3.
• the cytotoxic agent can be saporin or a biologically active variant thereof.
• the one or more MGS peptides can be SEQ ID NO: 3 and the cytotoxic agent can be saporin.
• the compositions can comprise one or more MGS peptides, for example, in some aspects, the composition can comprise, one, two, three, four or five MGS peptides.
• the one or more MGS peptides can form a tetrameric scaffold protein.
• the one or more MGS peptides disclosed herein can be truncated.
• the one or more MGS peptides can be modified.
• the one or more MGS peptides can acetylated on the N-terminus.
• the one or more MGS peptides can be chemically conjugated to the cytotoxic agent.
• the chemical conjugate can be polyethylene glycol (PEG).
• the number of PEG units can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more.
• the number of PEG units can be of sufficient length to separate the one or more MGS peptides from the cytotoxic agent to prevent any steric interference between the one or more MGS peptides and the cytotoxic agent.
• compositions comprising a chemical conjugate, wherein the chemical conjugate is PEG and the PEG comprises eleven PEG units.
• the one or more MGS peptides comprise SEQ ID NO: 3, wherein SEQ ID NO: 3 can be acetylated on the N-terminus and can be chemically conjugated to PEG; and the cytotoxic agent can be saporin, wherein the saporin can be covalently attached to PEG.
• Cytotoxic Agents A wide variety of toxic (e.g., cytotoxic) agents can be included in the disclosed compositions.
• the cytotoxic agents can be covalently conjugated or form a fusion protein with one or more of the MGS peptides disclosed herein.
• the cytotoxic agent can be a protein.
• the cytotoxic agent can be a bacteria or plant toxin.
• the cytotoxic agent can be a plant toxin.
• the cytotoxic agent can be saporin or a biological variant thereof.
• the cytotoxic agent can be modified.
• the cytotoxic agent is a fragment of a bacterial or plant toxin.
• the methods of making the conjugates or fusion proteins are known to one of ordinary skill in the art and can be carried out using known techniques.
• the MGS peptides are conjugated to a cytotoxic agent (e.g., saporin) using polyethylene glycol. It is within the skill of an ordinary person in the art to chemically conjugate the MGS peptides disclosed herein with the optimal PEG units.
• compositions comprising one or more molecular guidance system (MGS) peptides and a label.
• MMS molecular guidance system
• the compositions disclosed herein can include detectable labels.
• detectable labels can include, but are not limited to, a tag sequence designed for detection (e.g., purification or localization) of an expressed polypeptide or sequence.
• Tag sequences include, for example, green fluorescent protein, glutathione S -transferase, poly histi dine, c-myc, hemagglutinin, or FlagTM tag, and can be fused with an encoded nucleic acid.
• detectable labels can include, but are not limited to, a fluorescent agent, an enzymatic label, or a radioisotope.
• compositions comprising one or more of the compositions disclosed herein and a pharmaceutical acceptable carrier described above.
• the MSG peptide can be SEQ ID NO: 3 and the cytotoxic agent can be a saporin or a biological variant thereof and the pharmaceutical composition is formulated for intravenous administration.
• the compositions of the present disclosure also contain a therapeutically effective amount of the cytotoxic agent as described herein.
• compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration.
• excipient means any compound or substance, including those that can also be referred to as “carriers” or “diluents.”
• Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.
• compositions as disclosed herein can be prepared for oral or parenteral administration.
• Pharmaceutical compositions prepared for parenteral administration can be prepared for parenteral administration.
• compositions can be prepared for parenteral administration that includes fusion proteins dissolved or suspended in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
• an aqueous carrier such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
• compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like).
• a binder or filler e.g., for the formulation of a tablet, a capsule, and the like.
• the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.
• compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered.
• Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration.
• compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8).
• the resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above- mentioned agent or agents, such as in a sealed package of tablets or capsules.
• the composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
• MGS molecular guidance system
• cytotoxic agent can be saporin or a biologically variant thereof.
• the method can comprise administering one or more MGS peptides conjugated to a cytotoxic agent.
• the cytotoxic agent can target an intracellular target.
• the intracellular target can be a lysosome, golgi apparatus, endoplasmic reticulum, cytoplasm, or nucleus.
• skilled person can determine an efficacious dose, an efficacious schedule, or an efficacious route of administration for a disclosed composition or a disclosed fusion protein or a disclosed fusion protein so as to induce target an intracellular target such that the intracellular target can be inactivated.
• the composition, conjugate or fusion protein described herein can be combined with one or more additional therapies.
• the composition, conjugate or fusion protein can be administered alone or in combination with other biologically active agents into compositions suitable for administration to a subject.
• methods directed to treating subjects with cancer or at risk for developing cancer the composition, conjugate or fusion protein disclosed herein can be combined with, for example, therapeutically effective amount of radiation therapy, immunotherapy or chemotherapy or a combination thereof.
• the combined therapy can be administered as a co-formulation, or separately. When administered separately, the combined therapy can be administered simultaneously or sequentially.
• the formulations can be made using methods routine in the art.
• compositions described above can be formulated to include a therapeutically effective amount of a composition, conjugate or fusion protein as disclosed herein.
• Therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to one or more autoimmune diseases or where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to cancer.
• compositions described herein can be administered to the subject (e.g., a human subject or human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease.
• the subject is a human subject.
• compositions are administered to a subject (e.g., a human subject) already with or diagnosed with an autoimmune disease in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences.
• An amount adequate to accomplish this is defined as a "therapeutically effective amount.”
• a therapeutically effective amount of a pharmaceutical composition can be an amount that achieves a cure, but that outcome is only one among several that can be achieved.
• a therapeutically effective amount includes amounts that provide a treatment in which the onset or progression of the cancer is delayed, hindered, or prevented, or the autoimmune disease or a symptom of the autoimmune disease is ameliorated.
• One or more of the symptoms can be less severe. Recovery can be accelerated in an individual who has been treated.
• the cancer can be a primary, secondary, refractory or relapsing tumor.
• the cancer can be lung cancer, breast cancer, colorectal cancer, ovarian cancer or pancreatic cancer.
• Amounts effective for this use can depend on the severity of the cancer and the weight and general state and health of the subject. Suitable regimes for initial administration and booster administrations are typified by an initial administration followed by repeated doses at one or more hourly, daily, weekly, or monthly intervals by a subsequent
• compositions disclosed herein can be administered to a mammal as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, or once a month).
• a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, or once a month).
• continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are also within the scope of the present disclosure.
• the therapeutically effective amount of the toxins (or cytotoxic agents) present within the compositions described herein and used in the methods as disclosed herein applied to mammals (e.g., humans) can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, and other general conditions (as mentioned above). Because the compositions, conjugates and fusion proteins of the present disclosure can be stable in serum and the bloodstream and in some cases more specific, the dosage of the compositions, conjugates and fusion proteins including any individual component can be lower (or higher) than an effective dose of any of the individual components when unbound. Accordingly, in some aspects, the toxin administered can have an increased efficacy or reduced side effects when administered as part of a conjugate or fusion protein as compared to when the toxin is administered alone or not as part of a conjugate or fusion protein.
• vectors comprising the nucleic acid sequence that encodes for one or more of the disclosed compositions.
• the vector comprises only a nucleic acid sequence capable of encoding one or more of the disclosed MGS peptides.
• kits comprising one or more of the disclosed compositions.
• kits comprise a MGS peptide and a cytotoxic agent and instructions for conjugation.
• kits comprise a cell line comprising the nucleic acid sequence that encodes one or more of the MGS peptides.
• Example 1 HCC15.2 shows specificity to and internalizes in cancer cells
• FIG. 1 shows that the MGS peptide, HCC15.2, accumulates in cancer cells and is specific compared to control.
• HCC15.2 binding was analyzed by flow cytometry analysis (see, FIG. 1). The HCC15.2 does not bind to normal HBEC's but binds to the LC line H1299. The binding was sequence dependent and not specific for a single histopathological class (see, FIG. 2).
• MGS peptides were synthesized using standard solid-phase FMOC techniques on a Prelude synthesizer (Protein Technologies Inc.). MGS peptides were purified via reverse phase HPLC on a C18 prep column to >95% purity, and verified by MALDI-TOF.
• the optimized HCC15.2 Peptide sequence is Ac-FHAVPQSFYT-PEGl 1 -Biotin and has a molecular weight of 2394 Da, and Ac-FHAVPQSFYT-PEGl 1-C with a molecular weight of 1940 Da.
• Tetrameric HCC15.2 was synthesized via previously published protocols. Stock solutions were made in PBS pH 7.4 and concentration determined by absorbance at 280 nM on a Nanodrop 2000 (Thermo Fisher Scientific).
• cells are treated with a peptide (e.g., a MGS peptide) that has a dye on it for one hour. If the peptide enters the cell(s), the dye will also enter the cell(s) (see, FIG. 11).
• a peptide e.g., a MGS peptide
• the dye will also enter the cell(s) (see, FIG. 11).
• the cells are washed and placed in a tube to run flow cytometry.
• the flow cytometer can measure the brightness of 10,000 or more individual cells.
• cells are treated with a peptide (e.g., a MGS peptide) that has a dye on it for one hour. If the peptide enters the cell(s), the dye will also enter the cell(s) (see, FIG. 1).
• the cells are washed and fixed to glass plate. Other parts of the cell (e.g., cell membrane, organelles or nucleus) are stained. Fluorescent microscopy is performed to assess the dyes in the cells. Confocal microscopy is used to take a series of sliced images that can be used to build a 3D picture (like an MRI).
• HCC15.2 can be internalized and this internalization is receptor mediated (see, FIG. 3). More specifically, HCC15.2 binds to a specific subset of NSCLC and other tumor cells (see, FIG. 12). Phage blocking was used as a control. HCC15.2 was internalized by a specific panel of cell lines and not others. The results also show that the internalization mediated by a receptor was sequence specific.
• Treating with trypsin 2 min before adding HCC15.2 resulted in no intnernalization. Also, HCC15.2 does not internalize at 4° C.
• FIG. 4 shows that the tetramerization of HCC15.2 does not significantly improve receptor mediated internalization. This result is surprising as most peptides studied and identified through the FOX3 molecular guidance system had non-additive increase in binding/internalization.
• FIG. 5 and FIG. 15 show that the truncations from N- and C-Terminal ends of the HCC15.2 peptide reveal minimal binding sequence.
• FIG. 6 shows that acetylation improves binding of the truncated peptide.
• Example 4 Peptide co-localizes with the lysosome.
• FIG. 7 shows that the MGS peptide co-localizes with the lysosome and accumulates in the lysosome over time (see, FIG. 8 and FIG. 16).
• Example 5 HCC15.2 conjugated to saporin.
• FIG. 22 A cancer cell killing assay was performed (see, FIG. 22). Cancer cells were put into a dish. After 24 hours, cells were given different doses of peptide-saporin for 1 hour. Peptide-saporin was washed off and cells were left for 72 more hours. Assay to measure live cells performed.
• FIG. 21 shows that HCC15.2 targets saporin to cancer cells in vitro. These results demonstrate that saporin did not enter the cell by itself, however, saporin did enter the cell when conjugated to a required MGS peptide (e.g., HCC15.2). The results also show that HCC15.2-saporin lowers off target effects and escapes intracellular vesicles.
• FIG. 10 shows that HCC15.2 conjugated to saporin reduces tumor growth in a human NSCLC xenograft model compared to free-saporin.
• Flow cytometry is carried out to measure KD.
• Cells are treated with increasing concentrations of peptide-dye for one hour.
• Cells are washed and placed in a tube to run flow cytometry.
• the flow cytometer can measure brightness of 10,000 or more individual cells. Each concentration is quantified and plotted (see, FIGS. 13 and 14).
• the curves are mathematically fitted to data and the halfway point is KD (e.g., a measure of how good the binding is).
• Example 7 HCC 15.2- targeted NIR dye accumulates into tumors in vivo.
• Example 8 Further characterization of the H1299.3 peptide.
• a modified MGS peptide (SEQ ID NO: 31) that fuses amino acids 1-5 and 11-20 of the original MGS peptide: LQWRRNFGVWARYRL (SEQ ID NO: 31) was assessed.
• This MGS peptide maintains its cancer specificity and increases its ability to traffic to the autophagosome. Acetylation protects degradation in vivo and improves solubility.
• This MGS peptide hones to a NSCLC tumor in an animal (see, FIG. 27). The data show that no tumor targeting was observed for the nonacetylated version, This MGS peptide (e.g., the modified 15-mer) has >2-fold reduction in heart, lung and kidney tumors (see, FIG. 27). 1299.3 Ac-15mer cell binding results are shown in FIG. 28.
• Example 9 In vitro delivery of saporin with other peptides.
• FIG. 29 shows the IC50 in various cell lines of the administration of saporin conjugated or linked to select MGS peptides.

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