EP1943350A2 - Latent procytotoxins and uses thereof - Google Patents

Latent procytotoxins and uses thereof

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Publication number
EP1943350A2
EP1943350A2 EP06825387A EP06825387A EP1943350A2 EP 1943350 A2 EP1943350 A2 EP 1943350A2 EP 06825387 A EP06825387 A EP 06825387A EP 06825387 A EP06825387 A EP 06825387A EP 1943350 A2 EP1943350 A2 EP 1943350A2
Authority
EP
European Patent Office
Prior art keywords
peptide
cancer
cleavage site
cytolytic
procytotoxin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06825387A
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German (de)
French (fr)
Other versions
EP1943350A4 (en
Inventor
Thomas E. Wagner
Xianzhang Yu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Greenville Hospital System
Original Assignee
Greenville Hospital System
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Filing date
Publication date
Application filed by Greenville Hospital System filed Critical Greenville Hospital System
Publication of EP1943350A2 publication Critical patent/EP1943350A2/en
Publication of EP1943350A4 publication Critical patent/EP1943350A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • This invention relates to methods and compositions for selectively destroying a target cell. Specifically, the invention relates to methods for making and using a procytotoxin that can be converted to a cytotoxin in a target-cell specific manner, such that a target cell is destroyed by the activated cytotoxin.
  • chemotherapeutic agents are also toxic to normal cells, the dose of cytotoxic drug is adjusted to the limits of tolerance to achieve the maximum destruction of malignant cells, and the interval between doses must be such that the rate of tumor re-growth does not exceed tumor killing. Accordingly, in order to achieve increased efficiency with reduced side effects, the chemotherapeutic agents should have high target-cell specificity and high target-cell toxicity or potency. In spite of considerable research into therapies for cancer, currently available treatment methods are ineffective in a significant percentage of cases. 5. Accordingly, there is a need for improved cancer treatment and more methods that are not dependent upon the cell cycle of the cancer cell. The present invention fulfills these needs and further provides other related advantages.
  • This invention relates to methods and compositions for selectively destroying a target cell. Specifically, the invention relates to methods for making and vising a procytotoxin (where the cytotoxin is maintained in an inactive form) that can be converted to a cytotoxin in a target-cell specific manner, such that a target cell is destroyed by the activated cytotoxin.
  • a procytotoxin is provided which typically is made up of, for example, a cytotoxic agent bound to a latency associated peptide (LAP) via a peptide bond, and the peptide bond is susceptible to cleavage by a tumor specific protease.
  • LAP latency associated peptide
  • the procytotoxin can be described as V-Y-X, where V is a cytoxic agent, Y is a proteolytic cleavage site and Y is an LAP.
  • the cytotoxic agent can be, for example a cytotoxic peptide.
  • the peptide bond can be a proteolytic cleavage site.
  • the procytotoxin of the present invention can also have a targeting molecule linked to the N- and/or C-terminus of the cytotoxic peptide.
  • a cytotoxin is modified according to the invention to render it to a non-toxic form, known as a procytotoxin.
  • a procytotoxin is a cytotoxic agent that can be rendered non-toxic by providing a protective layer around the cytotoxic agent, hereinafter referred to as "caging".
  • the LAP can provide a protective "cage” around the pharmaceutically active agent thereby shielding it and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
  • the procytotoxin can also be a cytotoxic peptide that is rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic.
  • the procytotoxin can comprise a cytotoxic peptide bound to a LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease.
  • the LAP hinders the cytotoxic peptide from forming an active conformation.
  • a tumor specific protease for example resident in the tumor cell membrane or extracellular matrix, cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane.
  • a method for making a procytotoxin comprising modifying a cytotoxic peptide to include a LAP, is described.
  • compositions that contain a disclosed procytotoxin and a pharmaceutically suitable excipient.
  • methodology is provided for selectively destroying a target cell.
  • the disclosed approaches typically entail contacting the target cell with a procytotoxin, which has a cytotoxic agent bound via a peptide bond to an LAP, wherein the peptide bond is susceptible to cleavage by a target specific protease.
  • Target cells can be cells that are involved in the microvasculature surrounding cancer cells and cancer cells.
  • a method of treating cancer in a patient comprises administering to a patient a therapeutically effective amount of a procytotoxin.
  • procytotoxins can be based on an amoebopore, melittin, or a cytolytic peptide derived therefrom.
  • the method comprises covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin.
  • Figure 1 shows the detection of hLAP-MMP2-Melittin in transfected CHO cells.
  • 30 ug [search for ug/ul througout] of LAP CHO lystate (hLAP-MMP2-Melittin transfected) and CHO lysate (Not Transfected) was analyzed via SDS PAGE followed by western blotting with hLAP antiserum. 50ng of hLAP was used as a positive control.
  • Figure 2 shows methotrexate induction of LAP-MMP2-Melittin production in transfected CHO cells.
  • CHO cells were transfected with LAP-MMP2-Melittin plasmid DNA in the presence of normal transfection media.
  • Upon transfection cells were cultured in media containing 10% FBS and 5OnM MTX. The amount of FBS was gradually decreased in the media to 1 %.
  • Cells were then induced to produce protein by increasing the amount of MTX in the media. Lysate was obtained from cells at different MTX concentrations and analyzed via SDS PAGE followed by western blotting with h LAP antiserum.
  • FIG. 15 shows analysis of lysate and supernatant from hLAP-MMP2-Melittin transfected CHO cells under nondenaturing conditions.
  • hLAP fusion protein hLAP-MMP2- Melittin
  • FIG. 30 ug of fusion protein lysate was analyzed and 50ng of hLAP was used as a positive control. All samples were analyzed via Native PAGE followed by western blotting with hLAP antiserum.
  • Figure 4 shows analysis of partially purified hLAP-MMP2-Melittin fusion protein.
  • Purified hLAP-MMP2-Melittin Fusion protein was analyzed via SDS PAGE followed by western blotting with hLAP antiserum.
  • the fusion protein was partially purified via size exclusion chromatography on the Waters HPLC system.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment 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,” it will be understood that the particular value forms another embodiment. 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.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein.
  • a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • contacting the target cell with a procytotoxin means bringing the procytotoxin and target cell into close enough proximity with one another such that the a protease expressed by the target cell or resident in extraceullar matrix can cleave the proteolytic cleavage site of the procytotoxin, thus releasing the LAP from the cytotoxin and thereby allowing the cytotoxin to become active and exert its function on the target cell.
  • RGD refers not only to the peptide sequence Arg-Gly-Asp, it refers genetically to the class of minimal or core peptide sequences that mediate specific interaction with integrins.
  • an "RGD targeting sequence” encompasses the entire genus of integrin-binding domains.
  • NGR refers not only to the peptide sequence Asn-Gly-Arg, it refers genetically to the class of minimal or core peptide sequences that mediate specific interaction with integrin signal sequences that can be found on cells of the vasculature.
  • a NGR targeting sequence can be used to bind certain integrins and can serve as a useful targeting molecule to endothelial cells and other cells of the neo-vasculature.
  • an "NGR targeting sequence” encompasses the entire genus of integrin-binding domains.
  • the invention relates to methods for making and using a procytotoxin that can be converted to a cytotoxin at the site of a tumor-cell, such that a tumor cell is destroyed by the activated cytotoxin.
  • a procytotoxin is provided which typically is made up of a cytotoxic agent bound to a latency associated peptide (LAP) via a peptide bond, and the peptide bond is susceptible to cleavage by a tumor specific protease.
  • LAP latency associated peptide
  • the procytotoxin can be described as V-Y-X, where V is a cytoxic peptided, Y is a proteolytic cleavage site and Y is an LAP.
  • the cytotoxic agent can be a cytotoxic peptide.
  • the peptide bond can be a proteolytic cleavage site.
  • the procytotoxin of the present invention can also have a targeting molecule linked to the N- and/or C-terminus of the cytotoxic peptide.
  • Cytotoxic peptides can be cytolytic peptides.
  • Cytotoxins can include, but are not limited to: type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter ococcus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic
  • type I bacterial exotoxins can be used as a cytotoxin.
  • a type I bacterial exotoxin can be fused with a tumor cell membrane or tumor microenviroment targeting domain.
  • Type II bacterial exotoxins can also be used as a cytotoxin.
  • type III bacterial exotoxins can either retain its own trans-membrane domin or it can be fused with a different domain such as TAT. 36.
  • Examples of procytotoxins have the following structures: (1) Gly-Ile-Gly-Ala-
  • the LAP can also be the precursor peptide of human TGF ⁇ -1, 2 or 3 (SEQ ID NOS: 23- 25).
  • the LAP can also be the precursor peptide of chicken TGF ⁇ -4 (SEQ ID NO: 26).
  • the LAP can also be the precursor peptide of frog TGF ⁇ -5 (SEQ ID NO: 27).
  • the proteolytic cleavage site can be a MMP cleavage site, a PSA cleavage site or a GGH cleavage site.
  • the MMP proteolytic cleavage site can be a MMP 1, 2,
  • the MMP proteolytic cleavage site can also be any of the MMP proteolytic cleavage sites listed in SEQ ID NOS: 28-100.
  • proteolytic cleavage site can be a PSA cleavage site with the sequence described by Volkel et al. (Engineering of human coagulation factor X variants activated by prostate-specific antigen, Molecular Biotechnology, January 2005, vol. 29, no. 1, pp. 19-30(12))
  • the procytotoxin can further contain a targeting molecule.
  • the targeting molecule can interact, bind, attach, combine, join, connect to, or with, a target cell or tissue. More specifically, the targeting molecule can be a neovascular targeting sequence of an anti-fibronectin ED-B antibody.
  • the targeting molecule can be an RGD or NGR targeting sequence as described below. Any molecule that can target a specific tissue can be used as the targeting molecule of the present procytotoxin.
  • the targeting molecule can be an antibody that interacts with human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/pmell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM, alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm's tumor antigen
  • Procytotoxins can also be charge neutralized in addition to comprising steric inhibitors of the cytotoxin's activity.
  • steric inhibitors of the cytotoxin's activity For example, provided is [Gly-He-Gly-Ala-Val-Leu-
  • a cytotoxin is modified according to the invention to render it to a non-toxic form, known as a procytotoxin.
  • the procytotoxin is a cytotoxic peptide that can be rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic.
  • the procytotoxin can, alternatively, be a cytotoxic peptide that can be rendered non-toxic by providing a protective layer or shell around the cytotoxic peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
  • the procytotoxin comprises a cytotoxic peptide bound to a LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease.
  • the LAP hinders the cytotoxic peptide from forming an active conformation.
  • a tumor specific protease cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane.
  • a method for making a procytotoxin comprising modifying a cytotoxic peptide to include a LAP, is also described. 43.
  • a LAP may be added to either the N-terminus and/or C-terminus of the cytotoxic peptide to hinder the cytotoxic peptide from forming a conformation which renders the peptide toxic, therefore the cytotoxic peptide is rendered non-toxic.
  • a LAP can also be added to either the N- terminus and/or C-terminus of the cytotoxic peptide, such that the LAP forms a protective shell around the cytotoxic peptide peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
  • the LAP can also sterically preventthe alpha-helical structure of the cytotoxin from forming.
  • the cytotoxic peptide can be modified to include negatively charged amino acids, thereby preventing the toxic pore conformation from forming.
  • the procytotoxin of the present invention comprises a latency associated peptide and a cytotoxic agent, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic agent.
  • the cytotoxic agent can be a cytotoxic peptide or a cytolytic peptide, as described above.
  • the cytotoxic agent can be a melittin, a melittin analog, or a melittin derivative.
  • the procytotoxin of the present invention comprises the following structure:: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser- Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-[Y]-(X) wherein (X) is a LAP and [Y] is a proteolytic cleavage site that can be oriented in either direction.
  • the LAP can be selected from the group consisting of the precursor peptide of TGF ⁇ -1, 2, 3, 4, or 5.
  • the LAP can also be the precursor peptide of human TGF ⁇ -1, 2 or 3 (SEQ ID NOS: 23-25).
  • the LAP can also be the precursor peptide of chicken TGF ⁇ -4 (SEQ ID NO: 26).
  • the LAP can also be the precursor peptide of frog TGF ⁇ -5 (SEQ ID NO: 27).
  • the proteolytic cleavage site can be a MMP cleavage site, a PSA cleavage site or a GGH cleavage site.
  • the MMP proteolytic cleavage site can be a MMPl, 2, 3, 7, 8, 9, or 10 cleavage site.
  • the MMP cleavage site is the MMP2 cleavage site. Cleavage by a target specific protease at [Y] may yield a melittin peptide with a few additional amino acids on the C-terminus which should not interfere with pore formation.
  • a targeting molecule that adds an additional measure of selectivity.
  • the targeting molecule may direct the procytotoxin in or around the cell, as well as act as a LAP, maintaining the cytolytic peptide in its inactive conformation.
  • a targeting molecule of the instant invention can be selected from the group consisting of a signal sequence or an antibody.
  • the targeting molecule can be an RGD targeting sequence.
  • charge determinants are also important. Where the positive charge of the pore forming peptide is involved in its cytolytic activity, eliminating/neutralizing the charge renders the peptide non-toxic.
  • the procytotoxic peptides of this invention maybe chemically synthesized by standard solid phase procedures using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide.
  • a combination of manual and automated (e.g., APPLIED BIOSYSTEM. 430A) solid phase techniques can be used to synthesize the novel peptides of this invention.
  • APPLIED BIOSYSTEM. 430A e.g., APPLIED BIOSYSTEM. 430A
  • the in vivo stability of the procytotoxin of the invention can be improved by adding a D-amino acid to the N- or C-terminus, whichever does not have a ⁇ -linked glutamic acid residue.
  • Some in vivo instability might be advantageous because it could decrease the chance of possible adverse side effects that might arise once the procytotoxin is converted to cytotoxin. This procedure is particularly useful with products of the invention which are employed under conditions, parenteral or oral, where they will be subject to hydrolysis by naturally occurring enzymes before they perform their desired physiological function.
  • Cytotoxic Peptides 51 Many naturally occurring and synthetic cytotoxic peptides are known in the art.
  • the cytotoxic peptide is a cytolytic peptide.
  • Cytolytic peptides also known as pore-forming or channel-forming peptides, typically disrupt cell membranes, causing cell lysis and death upon contact.
  • Many naturally occurring cytolytic peptides from microorganisms, insects and higher animals are generally known. They often are called hemolysins because they lyse red blood cells as well as other eukaryotic cells.
  • toxins include type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I and other cytolysins of sea anemone, aerolysin, amatoxins, amoebapores, amoebapore homologs from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter ococcus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxins, enterotoxin o ⁇ Aeromonas hydwphila, esculentin, granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomy
  • cytolytic peptides include D,L- ⁇ -amino acid cyclic peptides. Fernandez-Lopez et al., Nature, 412:452-455 (2001).
  • type I bacterial exotoxins can be used as a cytotoxin.
  • a type I bacterial exotoxin can be fused with a tumor cell membrane or tumor microenviroment targeting domain.
  • Type II bacterial exotoxins can also be used as a cytotoxin.
  • type III bacterial exotoxins can either retain its own trans-membrane domin or it can be fused with a different domain such as TAT.
  • the cytolytic peptide can be a pore-forming or channel-forming cytolytic peptide.
  • Many cytolytic peptides are pore-forming toxins, belonging to a group of cytotoxins that associate with cell membranes, either nonspecifically or to specific receptors, and form transmembrane pores of discrete size.
  • Most toxic pore-forming peptides employ common features for their cell lysis activity. For example, a great number of these toxins lyse cells through a "barrel-stave" mechanism, in which monomers of the toxin bind to and insert into the target membrane and then aggregate like barrel staves surrounding a central, water filled pore. This pore causes rapid and irreversible electrolyte imbalance of the target cell leading to its destruction.
  • cytotoxic peptide can be a cytolytic, linear ⁇ -helical peptide. Generally these cytotoxic peptides, in their native form, will have a net positive charge, which contributes to their cytolytic activity.
  • the cytolytic peptide can be melittin or an analog or derivative thereof.
  • Melittin is isolated from bee venom and is a 26 amino acid amphiphilic alpha-helix (Blondelle et al., (1991) Biochemistry 30: 4671-4678; Dempsey et al., (1991) FEBS Lett. 281: 240-244.)
  • the amino acid sequence of melittin is shown in Table 1 and SEQ ID NO: 6.
  • Residues 1-20 are predominantly hydrophobic and residues 21 to 26 are hydrophilic and basic.
  • Melittin has antibiotic activity, but in mammals it is lytic for leukocytes, red blood cells and a wide variety of other cells.
  • Another pore-forming peptide can be amoebapore, a 77-residue pore-forming peptide from the amoebae Entamoeba histolytica (Young et al, (1982) J. Exp. Med. 156: 1677; Lynch et al, (1982) EMBO J 7: 801; Young & Cohn, (1985) J. Cell Biol. 29: 299; Rosenberg etal, (1989) Molec. Biochem. Parasit. 33: 237; Jansson et al., (1994) Science 263: 1440).
  • the amoebapore peptide is stabilized by three disulfide bonds and contains four mostly amphipathic alpha-helical structures.
  • the third amphipathic helical structure (helix 3) retains the cytolytic activity similar to the wild type peptide.
  • a synthetic peptide based on the sequence of its third amphipathic alpha helix has recently been shown to have cytolytic activity for nucleated cells at high concentrations (10-100 ⁇ M) (Leippe et ah, (1994) Proc. Natl. Acad. Sci.
  • the cytotoxin is an amoebapore derivative listed in Table 1 : NKb-Gly-Phe-Ile-Ala-Thr-Leu-Cys- Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-COOH.
  • cytolytic peptides may be ' modified or derivatized to produce analogs and derivatives which retain, or even exhibit enhanced, cytolytic activities.
  • Andra et al. disclose that shortened amoebapore analogs have enhanced antibacterial and cytolytic activities (FEBS Letters, 385 (1996), pp. 96-100, incorporated herein by reference in its entirety).
  • FEBS Letters, 385 (1996), pp. 96-100 incorporated herein by reference in its entirety.
  • 61 In designing such analogs or derivatives, the artisan will be informed by the foregoing discussion relating to the amphipathic alpha-helical structure and net positive charge that are implicated in the cytolytic activity.
  • cytotoxin analogs i.e., non-native forms never before known in nature, based on the observed homologies and known structure and properties of the native protein, to be used as a cytolytic peptide in accordance with the instant invention.
  • Modification and derivatization according to the instant invention include, but are not limited to, substitutions, additions or deletions that provide for functionally equivalent molecules. (Function may be assessed in accord with the working examples presented below.) Analogs and derivatives may also be made via modifications of side chains of amino acid residues of the cytotoxic peptides, for example, the analogs or derivatives can have enhanced or increased functional activity relative to the native protein or polypeptide.
  • homology and identity mean the same thing as similarity. Thus, for example, if the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not. 65.
  • variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
  • Nucleic acids 69 There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, melittin as well as any other proteins disclosed herein, as well as various functional nucleic acids.
  • the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell that the expressed mRNA will typically be made up of A, C, G, and U.
  • an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. Nucleotides and related molecules
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • An non-limiting example of a nucleotide would be 3'- AMP (3 '-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA).
  • Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • conjugates can be link other types of molecules to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • conjugates include but are not limited to lipid moieties such as a cholesterol moiety.
  • a Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
  • Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions can be made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions. TABLE 2: Amino Acid Abbreviations
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o- amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H.
  • variants of these and other proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to the stated sequence.
  • the variant retains the sole tryptophan residue at position 19 in the disclosed melittin sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. 85.
  • nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences.
  • each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 6 is set forth in SEQ ID NO: 5.
  • nucleic acid sequence that encodes the same protein sequence set forth in SEQ ID NO: 14 is set forth in SEQ ID NO: 13. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular procytotoxin from which that protein arises is also known and herein disclosed and described.
  • a particularly preferred non-peptide linkage is -CH 2 NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like. 90.
  • Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L-lysine
  • Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference) .
  • a latency associated peptide (LAP) according to the present invention is an object that, when attached to a lytic peptide, inactivates the cytotoxin by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic.
  • the LAP can vary in size. So long as the amino acids of the LAP cage, charge neutralize and/or create steric restriction of lytic peptide membrane insertion and organization into a pore, the peptide is a suitable LAP.
  • the LAP can be the precursor domain of TGF ⁇ or a sequence which is substantially identical thereto.
  • the LAP can also be a phage or phage particle. 95.
  • TGF ⁇ can be synthesized as a dimeric latent cytokine composed of an amino acid terminal latency associated protein (TGF ⁇ -LAP) and the active TGF ⁇ cytokine at its COOH terminal end (Roberts and Sporn, Peptide Growth Factors and their Receptors: Sporn, M B and Roberts, A B, Springer- Verlag, 419-472 (1996); Roth-Eicchorn et al., Hepatology, 28 1588-1596 (1998)).
  • the precursor peptide can contain a signal peptide (residues 1-29) necessary for protein secretion and guiding the molecule through the Golgi apparatus to become processed by proteolytic cleavage and glycosylation.
  • the TGF ⁇ -LAP domain can be separated from TGF ⁇ by proteolytic cleavage at arginines (277-278). Mature TGF ⁇ begins at alanine 279.
  • the TGF ⁇ -LAP in addition to preserving TGF ⁇ in a latent form, contains important residues necessary for the interaction with other molecules. Mutations in the TGF ⁇ -LAP domain have recently been associated with the autosomal dominant Camurati-Engelmann disease (Janssens et al., Nature Genetics, 26, 273:275 (2000). Cysteines 224 and 226 are important in the intermolecular disulphide bond between two TGF ⁇ -LAP proteins. Their mutation to serine renders the molecule "active" (Sanderson et al., Proc.
  • the RGD motif (245-247) facilitates the interaction with integrins (Munger et al., MoI, Biol, of the Cell, 9, 2627-2638 (1998; Derynck R, TIBS, 19, 548-553 (1994)).
  • the nucleic acid encoding TGF ⁇ is described in U.S. Pat. No. 5,801,231 and is herein incorporated by reference for its disclosure of the nucleic acid sequence encoding TGF ⁇ -LAP.
  • TGF ⁇ is secreted in a latent form consisting of TGF ⁇ subunit and TGF ⁇ - LAP propeptide dimers, covalently linked to latent TGF ⁇ -binding proteins (LTBPs).
  • LTBPs are also needed for the secretion and folding of TGF ⁇ (Miyazano et al., EMBO J. 10, 1091-1101 (1991); Miyazano et al., J. Biol. Chem. 267, 5668-5675 (1992); Eklov et al., Cancer Res. 53, 3193-3197 (1993)).
  • Cysteine 33 is important for the disulphide bridge with the third 8 cysteine-rich repeat of latent TGF ⁇ binding protein (LTBP) (Saharinen et al., The EMBO Journal, 15, 245-253 (1996). Modification of LTBP by enzymes such as thrombospondin (Schultz et al., The Journal of Biological Chemistry, 269, 26783-26788 (1994); Crawford et al., Cell, 93, 1159-1170 (1998)), transglutaminase (Nunes et al., J. Cell, Biol.
  • LTBP latent TGF ⁇ binding protein
  • the LAP of the present invention may comprise the precursor domain of TGF ⁇ , for example, the precursor peptide of TGF ⁇ -1, 2 or 3 (from human) (Derynck et al., Nature, 316, 701-705 (1985); De Martin et al., EMBO J. 6 3673-3677 (1987); Hanks et al., Proc. Natl. Acad. Sci. 85, 79-82 (1988); Derynck et al., EMBO J. 7, 3737-3743 (1988); Ten Dyke et al., Proc. Natl. Acad. Sci.
  • TGF ⁇ -4 from chicken
  • TGF ⁇ -5 from xenopus
  • the term "precursor domain” is defined as a sequence encoding a precursor peptide and does not include the sequence encoding the active TGF ⁇ protein
  • the amino acid sequence of the LAP can have at least 50% identity, using the default parameters of the BLAST computer program (Atschul et al., J. MoI. Biol. 215, 403-410 (1990) provided by HGMP (Human Genome Mapping Project), at the amino acid level, to the precursor domain of TGF ⁇ 1, 2, 3, 4 or 5 (Roberts and Sporn, Peptide Growth Factors and their Receptors: Sporn, M B and Roberts, A B, Springer- Verlag, Chapter 8, 422 (1996)).
  • BLAST computer program Altschul et al., J. MoI. Biol. 215, 403-410 (1990) provided by HGMP (Human Genome Mapping Project
  • the LAP can have at least 60%, 70%, 80%, 90%, or 95% identity, at the nucleic acid or amino acid level, to the LAP of TGF ⁇ 1, 2, 3, 4 or 5. In another embodiment the LAP can have up to or above 99% identity, at the nucleic acid or amino acid level, to the LAP of TGF ⁇ 1, 2, 3, 4 or 5.
  • the LAP can be seleted from the LAPs of SEQ ID NOS: 23-27.
  • the LAP may contain at least two, for example at least 4, 6, 8, 10 or 20 cysteine residues for the formation of disulphide bonds.
  • the LAP may provide a protective "cage" around the pharmaceutically active agent thereby shielding it and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
  • the LAP can comprise the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 1 or nucleotides 103-933 of SEQ ID NO: 3 or a sequence which has at least 50%, 60%, 70%, 80%. 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
  • the LAP can comprise the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 9 or nucleotides 97-928 of SEQ ID NO: 11 or a sequence which has at least 50%, 60%, 70%, 80%. 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
  • the LAP can be added to the N- or C-terminus, or both the N- and C- terminus, of the cytotoxic peptide.
  • the cytotoxic peptide can be a cytolytic peptide
  • the LAP can be added to the C-terminus of a cytolytic peptide.
  • the LAP may additionally comprise a targeting molecule, as discussed herein.
  • a charge neutralization component can be added to the procytotoxin where the procytotoxins further are charge neutralized, in addition to steric determinants.
  • a peptide comprising [Gly-Ile-Gly-Ala-Val-Leu- LysC ⁇ -Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-LysC ⁇ -Arg-LysC ⁇ -Arg- GIn-GIn-GIy-AIa-IIe-GIy-GIn-PrO]-(X), wherein (X) is the LAP as described herein, which is bound to the peptide marked in brackets via a peptide bond, wherein the peptide marked in brackets can be oriented in either direction, wherein the peptide bond is susceptible to cleavage by a tumor specific protease and wherein R is independently selected from the group consisting of the unmodified ⁇ -amino group of the adjacent lysine residue, [ ⁇ - ⁇ ]-Glu, [ ⁇ - ⁇ ]-Glu-[ ⁇ - ⁇ ]-(Glu) 1-3
  • a target specific protease as described herein is an enzyme associated with a target cell that cleaves a peptide bond of proteins.
  • a target cell as described herein, can be any cell whose lysis is desired.
  • a target cell could be a cancer cell, a bacterial cell, a neuronal cell, a bone marrow cell, a prostate cell, or an endothelial cell of the tumor neo-vasculature.
  • the targeting specific protease of the present invention may be typically expressed in tumors, but need not be exclusively expressed in tumors. Accordingly, the targeting specific protease may be preferentially expressed in tumor cells when compared to normal cells. Additionally, the targeting specific protease may be a protease expressed by a non-tumorigenic target cell, such as a bacterial cell, but is nevertheless preferentially expressed by a target cell when compared to a non-target cell. Accordingly, the procytotoxin as described herein is additionally useful as an antimicrobial or antibacterial agent. The procytotoxin as described herein is also additionally useful for lysing any target cell of interst, so long as the targeting specific protease is present in the target cell.
  • MMP Matrix Metalloproteinase
  • the targeting specific protease can be a matrix metalloprotease (MMP).
  • MMPs are associated with angiogenesis, as well as neo-vessel formation. Therefore, targeting the vasculature which supplies the tumors, as well as newly formed vessels, can serve to eliminate blood flow to tumors and can also prevent tumors from forming.
  • cytolytic peptides linked to an LAP by a sequence of amino acids recognized by MMP can be selectively released from the LAP in the presence of a targeting specific protease such as MMP.
  • MMP targeting specific protease
  • the cytotoxic peptide is then free to form its active conformation and lyse target cells.
  • PSA Prostate Specific Antigen
  • PSA prostate-specific antigen
  • PSA is the product of the KLK3 gene, and is expressed by the prostate and other tissues. It is expressed by healthy cells but abnormally high levels of PSA in the blood are found in men prostate disorders including prostate cancer and in women with breast cancer. PSA is an important tumor marker used in the diagnosis and monitoring of both prostate and breast cancer.
  • PSA is a 33 -kd protein consisting of a single-chain glycoprotein of 237 amino acid residues, 4 carbohydrate side chains, and multiple disulfide bonds.
  • PSA is homologous with the proteases of the kallikrein family.
  • PSA may be referred to as human glandular kallikrein-3 (hK-3) to distinguish PSA from human glandular kallikrein-2 (hK-2), another prostate cancer marker with which it shares 80% homology.
  • Human glandular kallikrein- 1 (hK-1) is a kallikrein that is found primarily in pancreas and renal tissue but shows 73% and 84% homology with PSA.
  • the complete gene encoding PSA has been sequenced and localized to chromosome 19. PSA can be found in prostate epithelial cells and in the seminal fluid. 114.
  • PSA gene transcription in the prostate is known to be regulated by androgens through the action of the AR (Riegman et al., MoI Endocrinol (1991); Schuur et al., J Biol Chem (1996); Cleutjens et al., MoI Endocrinol (1997); and Young et al., Cancer Res (1991)).
  • AR androgen response elements
  • PSA semenogelin
  • PSA insulin-like growth factor binding protein 3
  • protein C inhibitor protein C inhibitor
  • TGF- ⁇ transforming growth factor- ⁇
  • PTH-related peptide PTH-related peptide
  • PSA-ACT Ot 1 - antichymotrypsin
  • PSA- A2M ⁇ 2 -macroglobulin
  • free PSA Stenman et al., Cancer Res (1991) and Christensson et al., Eur J Biochem (1990)
  • PSMA Prostate Specific Membrane Antigen
  • PSMA prostate specific membrane antigen
  • PSMA prostate specific membrane antigen
  • Prostate specific membrane antigen a unique glutamate carboxypeptidase: a review of recent findings, The Prostate J. 1 : 15-26; Wright et al. (1995) Expression of Prostate Specific Membrane Antigen (PSMA) in normal benign and malignant prostate tissues, Urol. Oncol. 1 : 18-28; Lopes et ah, (1990) Immunohistochemical and pharmacokinetic characterization of the site-specific immunoconjugate CYT-356 derived from antiprostate monoclonal antibody 7El 1-C5, Cancer Res. 50: 623-6428; Troyer et al., Detection and characterization of the prostate- specific membrane antigen (PSMA) in tissue extracts and body fluids, Int. J. Cancer 62: 552-558).
  • the proteolytic domain of PSMA is located on the outside of the cell surface.
  • the procytotoxins of the invention without having to be internalized by the target cell, will have their ⁇ -glutamate residue(s) cleaved and removed.
  • the procytotoxins thus can be activated precisely at the desired site of action, the target cell, as long as a similar mechanism is present to remove the LAP.
  • This direct effect on the target cell increases the effectiveness of the prostate cancer cell killing.
  • the activated cytolytic peptide immediately inserts into the target cell, leaving it almost no chance of acting upon adjacent, non-target cells. In fact, it is possible that one end of the peptide is already inserted into the membrane before it is activated by PSMA.
  • the activated toxin is neutralized by the membrane therefore after the lysis of the target cell, the cytolytic peptides remain adsorbed in the membrane debris of the target cell and do not leak and harm non-target cells.
  • GGH Gamma-Glutamyl Hydrolase
  • GGH Gamma-glutamyl hydrolase
  • GGH EC 3.4.19.9
  • GGH is a central enzyme in the metabolism of folyl and antifolyl poly-gamma-glutamates (McGuire, J. J., and Coward, J. K. (1984) in Folates and Pterins (Blakely, R. L. , and Benkovic, S. L., eds), Vol. 1 , pp. 135-190, John Wiley & Sons, Inc., NY).
  • Folate is required as a cofactor by several enzymes in the de novo biosynthesis of DNA precursors and of several amino acids and is essential for normal cell growth and replication.
  • Antifolates such as methotrexate (MTX) have been the traditional treatment for many cancers over almost four decades (Chabner et al., J. Clin. Invest. 76, 907-912).
  • folates or antifolates When transported into the cell, they are converted to folylpoly-gamma- glutamates or antifolylpoly-gamma-glutamates through the sequential addition of glutamates by folylpolyglutamate synthetase (FPGS) (EC 6.3.2.17).
  • FPGS folylpolyglutamate synthetase
  • These poly-gamma-glutamates are retained intracellularlyand are generally better substrates or inhibitors than the corresponding monoglutamates for most of the folate-dependent enzymes (Galivan, J. (198O) Mo/.
  • GH catalyzes the removal of the gamma-linked polyglutamate chain, converting its substrates to folyl- or antifolyl-mono-gamma-glutamates, which are less well retained, and thus resulting in a reduction in the overall effectiveness of folates and antifolates.
  • FPGS and GH activities in the regulation of the extent of polyglutamylation of folates or antifolate drugs are well demonstrated.
  • Increased GH activity (Rhee, M. S., Wang, Y., Nair, M. G., and Galivan, J. (1993) Cancer Res. 53, 2227-2230; Yao, R., Rhee, M. S., and Galivan, J.
  • the proteolytic cleavage site may comprise any protease specific cleavage site.
  • the proteolytic cleavage site can include, but is not limited to, a matrix metalloproteinase (MMP) cleavage site, a PSA cleavage site, a PSMA cleavage site, or a GGH cleavage site.
  • MMP matrix metalloproteinase
  • the MMP cleavage site may comprise any amino acid sequence which is cleavable by a MMP.
  • the amino acid sequence of the MMP cleavage site may be encoded by nucleotides 832-852 of the procytotoxin shown in SEQ U) NO: 1 (melittin based) or nucleotides 832-852 of the procytotoxin shown in SEQ ID NO: 9 (amoebopore based) or a sequence of nucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
  • the nucleic acid sequence encoding the MMP cleavage site can comprise the minimum number of residues required for recognition and cleavage by MMP.
  • a MMP cleavage site may comprise a number of amino acid residues recognizable by MMP. Moreover, the amino acids of the MMP site may be linked by one or more peptide bonds which are cleavable, proteolytically, by MMP.
  • MMPs which may cleave the MMP site include, but are not limited to, MMPl, MMP2, MMP3, MMP7, MMP8, MMP9, MMPlO or MTl-MMP (Yu and Stamenkovic, Genes and Dev. 14, 163- 176 (2000); Nagase and Fields, Biopolymers, 40, 399-416 (1996); Massova et al., J. MoI. Model.
  • the proteolytic cleavage site of the present invention is cleaved at sites of tumor presence and tumor neo-vascular formation.
  • the proteolytic cleavage site of the present invention is a MMP cleavage site e.g any one or more of MMPl, MMP2, MMP3, MMP7, MMP8, MMP9 or MMPlO as shown in SEQ ID NOS: 28-100.
  • the cleavage site is a MMP2 cleavage site.
  • cytotoxic peptides of the instant invention linked to an LAP by a sequence of amino acids recognized by MMP can be selectively released from the LAP in the presence of a tumor specific protease such as MMP. The cytotoxic peptide is then free to form its active conformation and destroy the target cells.
  • the targeting molecule of the instant invention is an optional feature that presents an extra measure of selectivity.
  • the targeting molecule directs the procytotoxin to the target cell, where the procytotoxin is rendered toxic and selectively lyses its target. Additionally, the targeting molecule may act as an LAP of the cytolytic peptide. In this regard, the targeting molecule may charge neutralize or sterically inhibit a cytolytic peptide from pore formation.
  • the targeting molecule may be added to the N- or C-terminus, or both.
  • the targeting molecule is an antibody.
  • the antibody can be an anti-fibronectin ED-B antibody, thereby directing the procytotoxin to the extracellular matrix associated with neo-vessel formation.
  • Targeting molecules that target the neo-vasculature can also be added to the LAP. Molecules that target the neo-vasculature can easily be identified by screening phage display libraries. Any such peptide would be a suitable targeting molecule of the present invention.
  • Targeting molecules which bind specifically to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides bear the signal sequence based on Arg-Gly-Asp (RGD). Accordingly, sequences that bind certain integrins can serve as useful targeting molecules to endothelial cells and other cells of the neo-vasculature.
  • the targeting molecule can be an RGD targeting sequence.
  • Non-structural spacers may be a feature of the targeting molecule. Such spacers typically comprise glycine and/or proline residues. Lengths of these spacers can range from about one to about 5 amino acids. In addition, it is often preferable to physically constrain the targeting molecule by cyclization, which usually results in increased binding. This is usually accomplished by a pair of cysteine residues, flanking the RGD core at a distance of about 4 (having only RGD in between) to 10 amino acids from one another. For example, the pair of cysteine residues, flanking the RGD core can be at a distance of 7 amino acids from one another.
  • a typical targeting molecule would have the following structure:
  • X is zero to five amino acids and Y is a one or two amino acids, selected from cysteine, serine, threonine and methionine, hi a particularly useful embodiment, X is comprised of glycine residues, but optionally contains at least one, and typically one or two, free thiol- or amine-containing amino acids and/or a single hydrophobic amino acid.
  • Thiol- containing residues include methionine and cysteine; amine-containing residues include lysine and (at least one additional) arginine; and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine. 134.
  • Targeting molecules which bind specifically to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides bear the signal sequence based on Asn-Gly-Arg (NGR). Accordingly, sequences that bind certain integrins can serve as useful targeting molecules to endothelial cells and other cells of the neo-vasculature.
  • the targeting molecule can be an NGR targeting sequence.
  • Non-structural spacers may be a feature of the targeting molecule.
  • Such spacers typically comprise glycine and/or proline residues. Lengths of these spacers can range from about one to about 5 amino acids, hi addition, it is often preferable to physically constrain the targeting molecule by cyclization, which usually results in increased binding. This is usually accomplished by a pair of cysteine residues, flanking the NGR core at a distance of about 4 (having only NGR in between) to 10 amino acids from one another. For example, the pair of cysteine residues, flanking the NGR core can be at a distance of 7 amino acids from one another.
  • a typical targeting molecule would have the following structure: -XNGRYX- wherein X is zero to five amino acids and Y is a one or two amino acids, selected from cysteine, serine, threonine and methionine.
  • X is comprised of glycine residues, but optionally contains at least one, and typically one or two, free thiol- or amine-containing amino acids and/or a single hydrophobic amino acid.
  • Thiol- containing residues include methionine and cysteine; amine-containing residues include lysine and (at least one additional) arginine; and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine.
  • the targeting molecule can also be a molecule that interacts with a tumor antigen.
  • the tumor antigen can be selected from the list consisting of human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/ ⁇ mell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM, alpha- fetoprotein (AFP), COl 7-1 A, GA733, gp72, p
  • the targeting molecule can also be an aptamer or an antibody specific for the target.
  • the targeting molecule can also be an aptamer or an antibody specific for the target.
  • the methods of the present invention may utilize aptamers with unique or improved binding characteristics to a target that is unique to or over represented (as compared to a normal or non-target cell) in, around or on a cell of interest.
  • An "aptamer” as used herein refers to a nucleic acid that binds a target molecule through interactions or conformations other than those of nucleic acid annealing/hybridization described herein.
  • Methods for making and modifying aptamers, and assaying the binding of an aptamer to a target molecule may be assayed or screened for by any mechanism known to those of skill in the art (see for example, U.S. Pat. Nos. 6,111,095, 5,861,501, 5,840,867, 5,792,613, 5,780,610, 5,780,449, 5,756,291 5,631,146 and 5,582,981; as well as PCT Publication Nos. WO92/14843, WO91/19813, and WO92/05285, each of which is incorporated herein by reference).
  • Aptamers are single- or double-stranded DNA or single-stranded RNA molecules that recognize and bind to a desired target molecule by virtue of their shapes. See, e.g., PCT Publication Nos. WO92/14843, WO91/19813, and WO92/05285.
  • the SELEX procedure described in U.S. Pat. No. 5,270,163 to Gold et al., Tuerk et al. (1990) Science 249:505-510, Szostak et al. (1990) Nature 346:818-822 and Joyce (1989) Gene 82:83-87, can be used to select for RNA or DNA aptamers that are target-specific.
  • an oligonucleotide is constructed wherein an n-mer, preferably a random sequence tract of nucleotides thereby forming a "randomer pool" of oligonucleotides, is flanked by two polymerase chain reaction (PCR) primers.
  • PCR polymerase chain reaction
  • oligonucleotides which bind the target molecule are: (a) separated from those oligonucleotides which do not bind the target molecule using conventional methods such as filtration, centrifugation, chromatography, or the like; (b) dissociated from the target molecule; and (c) amplified using conventional PCR technology to form a ligand- enriched pool of oligonucleotides. Further rounds of binding, separation, dissociation and amplification are performed until an aptamer with the desired binding affinity, specificity or both is achieved. The final aptamer sequence identified can then be prepared chemically or by in vitro transcription.
  • the length of a random sequence tract can range from 20 to over 150 residues, and can be even longer if multiple, random oligonucleotides are combined into a single pool by ligation or other methods.
  • the number of individuals in a random sequence population is typically at least 10.sup.13 and can easily be over 10.sup.15. For most pools, this means that upwards of all possible 25-mers are present, and a proportionately smaller number of motifs longer than 25. Because of the redundancy of biological sequences, the sequence diversity of most random sequence pools likely rivals the sequence diversity of the Earth's biosphere.
  • RNA aptamers specifically interact with the prion protein PrP. J Virol 71(11): 8790-7; Convery, M. A., S. Rowsell, et al. (1998). "Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution.” Nat Struct Biol 5(2): 133-9; Homann, M. and H. U. Goringer (1999). "Combinatorial selection of high affinity RNA ligands to live African trypanosomes.” Nucleic Acids Res 27(9): 2006-14.). La particular, aptamers have been selected against a wide variety of proteins, including many nucleic acid binding proteins, such as T4 DNA polymerase (Tuerk, C. and L. Gold (1990).
  • RNA molecules that bind to and inhibit the active site of a tyrosine phosphatase J Biol Chem 273(23): 14309-14.
  • cytokines such as basic fibroblast growth factor (Jellinek, D., C. K. Lynott, et al. (1993).
  • High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding Proc Natl Acad Sci U S A 90(23): 11227- 31; Jellinek, D., L. S. Green, et al. (1995).
  • RNA epitope immunologically cross-reactive with a peptide As the combining sites of antibodies (Tsai, D. E., D. J. Kenan, et al. (1992). "In vitro selection of an RNA epitope immunologically cross-reactive with a peptide.” Proc Natl Acad Sci U S A 89(19): 8864-8) or the active sites of enzymes. (Tuerk, C, S. MacDougal, et al. (1992). "RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.” Proc Natl Acad Sci U S A 89(15): 6988-92.).
  • aptamers Almost all proteins have either surface pockets or basic patches (indeed, even proteins with negative pi's, such as T4 DNA polymerase, typically contain sites that can elicit aptamers). Most aptamer: target complexes have dissociation constants in the nanomolar range. Moreover, aptamers recognize their targets with high specificity, and can typically discriminate between protein targets that are highly homologous or differ by only a few amino acids. (Conrad, R., L. M. Keranen, et al. (1994). "Isozyme-specific inhibition of protein kinase C by RNA aptamers.” J Biol Chem 269(51): 32051-4; Eaton, B. E., L. Gold, et al. (1995).
  • antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class.
  • Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains.
  • L light
  • H heavy
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes.
  • IgA human immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR).
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., "Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1987)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • fragments of antibodies which maintain integrin binding activity are included within the meaning of the term “antibody or fragment thereof.”
  • an antibody or a fragment thereof that maintains alphaVbeta3 binding activity is included within the meaning of the term "antibody or fragment thereof.
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference. 148.
  • the antibodies are generated in other species and "humanized” for administration in humans.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, hi general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. 150.
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994).
  • Transgenic animals e.g., mice
  • J(H) antibody heavy chain joining region
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988).
  • a hybridoma method a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent can be selected from the list consisting of human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/pmell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV- 16, MUM, alpha-fetoprotein (AFP), CO 17- 1 A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm's
  • DNA-based immunization can be used, wherein DNA encoding a portion of integrin expressed as a fusion protein with human IgGl is injected into the host animal according to methods known in the art (e.g., Kilpatrick KE, et al. Gene gun delivered DNA- based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor. Hybridoma.
  • An alternate approach to immunizations with either purified protein or DNA is to use antigen expressed in baculovirus.
  • the advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems.
  • Use of this system involves expressing domains of an integrin antibody as fusion proteins.
  • the antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the integrin antibody nucleotide sequence. This results in the display of the foreign proteins on the surface of the virion. This method allows immunization with whole virus, eliminating the need for purification of target antigens. 156.
  • peripheral blood lymphocytes are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, "Monoclonal Antibodies: Principles and Practice” Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Immortalized cell lines can be those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • Other immortalized cell lines can be murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RJA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RJA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells can serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for integrins and another antigen- combining site having specificity for a different antigen.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988).
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab')2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.
  • the Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region.
  • the F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • Antibody fragments originally were produced as pairs of Fab 1 fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • An isolated immunogenically specific paratope or fragment of the antibody is also provided.
  • a specific immunogenic epitope of the antibody can be isolated from the whole antibody by chemical or mechanical disruption of the molecule.
  • the purified fragments thus obtained are tested to determine their immunogenicity and specificity by the methods taught herein, hnmunoreactive paratopes of the antibody, optionally, are synthesized directly.
  • An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the antibody amino acid sequence.
  • One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • Boc tert - butyloxycarbonoyl
  • a peptide or polypeptide corresponding to the antibody for example, can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • This native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (JLrB) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al.,
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
  • polypeptide fragments which have bioactivity.
  • the polypeptide fragments can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity.
  • amino or carboxy-terminal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays.
  • a fragment of an antibody comprises a modified antibody wherein at least one amino acid has been substituted for the naturally occurring amino acid at a specific position, and a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified antibody.
  • a modified antibody can be fused to a maltose binding protein, through either peptide chemistry or cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide.
  • the hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164.). Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well.
  • the fragments include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment.
  • ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • the nucleic acids can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B).
  • a Milligen or Beckman System lPlus DNA synthesizer for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B.
  • a particular advantage of the useful peptides of this invention is that they are readily synthesized by solid phase methods and a variety of combinations are possible to achieve specifically required results.
  • An advantage of the use of solid phase techniques is that the product can be directly synthesized with the C-terminus amidated or otherwise blocked, which is beneficial in forming the procytotoxins of the invention.
  • One method of producing the disclosed proteins is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • Boc tert -butyloxycarbonoyl
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett.
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)). 175.
  • Another method of producing the disclosed proteins, such as (melittin) SEQ ID NO: 6, is is to use recombinant DNA methods such as those described in U.S. Pat. No.
  • DNA encoding the the proteins can be readily isolated and sequenced using conventional procedures.
  • the hybridoma cells can serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce the protein, to obtain the synthesis of the protein in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce the protein, to obtain the synthesis of the protein in the recombinant host cells.
  • compositions 176 are also provided, in achieving this objective of the invention, are pharmaceutical compositions that, in general, contain a disclosed procytotoxin and a pharmaceutically suitable excipient
  • Another aspect of the present invention is a pharmaceutical composition comprising one or more procytotoxins of the invention and a pharmaceutically suitable carrier or excipient.
  • compositions of the present invention can be administered, alone, to a patient, it is also possible to administer the compositions in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • Parenteral administration of the composition, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is herein incorporated by reference. 180.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular target as described above as well as below.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). 182.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • compositions described herein can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, pills, powders, granules or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can also be used. 191.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the procytotoxins of the invention are amphoteric they may be utilized as free bases, as acid addition salts or as metal salts.
  • the salts must, of course, be pharmaceutically acceptable, and these will include metal salts particularly alkali and alkaline earth metal salts, suitably potassium or sodium salts.
  • a wide variety of pharmaceutically acceptable acid addition salts are available. These include those prepared from both organic and inorganic acids, for example, the pharmaceutically acceptable acid addition salt can be mineral acids. Typical acids which may be mentioned by way of example include citric, succinic, lactic, hydrochloric and hydrobromic acids. Such products are readily prepared by procedures well known to one skilled in the art. 193.
  • compositions of the present invention can also be modified to have a longer clearance rate and therefore increase bioavailability by protecting the composition from an immune response and other clearance mechanisms afforded by the subject.
  • WO 9/2802 discloses that PEGylated compounds exhibit reduced immunogenicity and antigenicity, and circulate in the bloodstream considerably longer than unconjugated proteins.
  • PEG (polyethyleneglycol) polymer chains can be attached to the prolactin variant/prolactin receptor antagonist by methods known in the art, such as by the PEGylation procedure described in Wang et al, Advanced Drug Delivery Reviews; 54 (2002) 547-570, which is incorporated herein by reference.
  • compositions described herein can also be modified with hydroxyethylstarch (HES).
  • HES hydroxyethylstarch
  • Methods for making HES-protein conjugates are known in the art. See, for example, EP 1398322, DE 2616086 and DE 2646854, which are incorporated herein by reference.
  • the cytolytic peptides will normally be the principal physiologically active ingredient.
  • inventive peptides may be formulated, however, with additional pharmacological agents for combination therapies. When used in treating cancer, for example, they may be formulated with compatible conventional chemotherapeutic agents.
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of a disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the efficacy of the therapeutic composition can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition disclosed herein is efficacious in treating or inhibiting tumor formation or tumor growth in a subject by observing that the composition reduces tumor formation or tumor growth or prevents a further increase in tumor formation or tumor growth. Efficacy of the administration of the disclosed composition may also be determined by measuring the level of tumor specific antigens present in the blood. Methods of measuring the level of tumor specific antigens are well known in the art.
  • compositions that inhibit tumor formation or tumor growth disclosed herein may be administered prophylactically to patients or subjects who are at risk for tumor formation or tumor growth.
  • compositions and methods can also be used for example as tools to isolate and test new drag candidates for a variety of cancers.
  • compositions and methods can also be used to study cell death.
  • the disclosed composition and methods can also be used to study cell expression of MMP in cells.
  • compositions and methods can also be used to study the effect of different cytotoxins on different cell types and also to study the ability of different enzymes to cleave different proteolytic cleavage sites.
  • Method of Selectively Destroying a Target Cell 204 Also disclosed are methods for selectively destroying a target cell.
  • the disclosed methods typically entail contacting the target cell with a procytotoxin disclosed herein.
  • the procytotoxin can have a cytotoxic peptide bound via a peptide bond to an LAP, wherein the peptide bond is susceptible to cleavage by a target specific protease.
  • Target cells can be cells that are involved in the microvasculature surrounding cancer cells and cancer cells.
  • the procytotoxin of the present invention typically is converted, and thereby activated, into a cytotoxic peptide in a target cell-specific manner by an activity associated with the target cell.
  • the target cell possesses a specific mechanism for converting the procytotoxin into a cytotoxin.
  • the activated cytotoxic peptide acts on and destroys the target cell in a selective manner.
  • the cell-associated activity can be a protease.
  • a method for selectively destroying a target cell comprising contacting the target cell with procytotoxin, wherein a proteolytic cleavage site is provided between a latency associated peptide and a cytotoxic peptide, is described.
  • the target cell can be a cancer cell, however any cell that comprises a cell specific protease may be a target.
  • the target cell can have a protease that selectively cleaves the proteolytic cleavage site to which the LAP is attached. Therefore, if the procytotoxin comprises an MMP cleavage site, a target cell with an MMP can cleave the proteolytic cleavage site and "activate" the cytolytic peptide, hi addition, the procytotoxin can comprise a targeting molecule.
  • the targeting sequences can also act as LAPs themselves, as well as direct a procytotoxin to the neo-vasculature and cytoskeletal elements surrounding the target cell.
  • the procytotoxin can comprise a targeting molecule selected from the group consisting of an RGD targeting sequence and an anti-fibronectin ED-B antibody.
  • the procytotoxin comprises a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is a cytolytic peptide, and wherein the proteolytic cleavage site is an MMP cleavage site, and wherein the LAP comprises the precursor peptide of TGF ⁇ -1, 2, 3, 4, or 5.
  • the procytotoxin can comprise a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is melittin, wherein the proteolytic cleavage site is an MMP2 cleavage site and wherein the LAP is the precursor peptide of TGF ⁇ -1.
  • the procytotoxin can comprise a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is an amoebopore, wherein the proteolytic cleavage site is an MMP2 cleavage site and wherein the LAP is the precursor peptide of TGF ⁇ -1.
  • a non-target cell which does not contain the MMP, is unable to activate the protoxin and is thus unaffected by the protoxin.
  • Therapeutic treatment of cancer using the instant cytolytic peptide-based procytotoxin is particularly advantageous because cytolytic peptides are known to be absorbed into the target cell membrane. Even after the target cell is lysed, therefore, the cytolytic peptides are prevented from acting on, and causing undesired destruction of, adjacent non-target cells. For example, see Leippe et al. (1991) Proc. Natl. Acad. Sci. 88: 7659-7663. Moreover, at least in the case where no extra stabilization steps (described above) are taken, it is noted that the instant medicaments are small peptides, which generally will have a fairly short half-life. Hence, even if small amounts of activated cytolytic peptide escapes the surface of the cancer cell, it should be reasonably short-lived and cause little, if any, destruction of non-target cells.
  • Method for Treating Cancer in a Patient 211 Further disclosed is a method of treating cancer in a patient, hi general, the method comprises administering to a patient a therapeutically effective amount of a procytotoxin.
  • procytotoxins can be based on an amoebopore, melittin or a cytolytic peptide derived therefrom.
  • the dosage of the procytotoxin might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day.
  • the dosage can be from 5 mg/kg to 10 mg/kg.
  • More than one procytotoxin may be administered, and combinations of procytotoxins may also be administered.
  • Optimal delivery routes, dosages and regimens for a given mammalian host can be readily ascertained by one skilled in the art. It will, of course, be appreciated that the actual dose used will vary according to the particular composition formulated, the particular compound used, the mode of application and the particular site, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease.
  • the disclosed compositions can be used to treat any solid tumor.
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer
  • a method for treating a cancer patient can comprise administering to a patient a therapeutically effective amount of a procytotoxin comprising a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and a cytotoxic peptide.
  • the cytotoxic peptide can be a pore-forming cytolytic peptide, as described above.
  • the cytolytic peptide can be a melittin, a melittin analog, or a melittin derivative.
  • the procytotoxin is a cytotoxic peptide that is rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic.
  • the procytotoxin comprises a cytotoxic peptide bound to an LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease.
  • the LAP of the instant invention hinders the cytotoxic peptide from forming an active conformation.
  • a tumor specific protease cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane.
  • a method of providing latency to a cytotoxin can comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin.
  • the cytotoxin can be a pore-forming cytolytic peptide, as described above.
  • the cytolytic peptide can be a melittin, a melittin analog, or a melittin derivative.
  • the proteolytic cleavage site can be an MMP cleavage site, and the LAP can comprise the precursor peptide of TGF ⁇ -1, 2, 3, 4, or 5.
  • a method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said cytotoxin is hindered from forming a conformation which renders the peptide toxic, therefore the cytotoxic peptide is rendered non-toxic. 220.
  • a method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide forms a protective shell around the cytotoxic peptide peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
  • This can be achieved by allowing the LAP to form a dimmer around the cytotoxic peptide, thus forming a protective shell, or cage, around the cytotoxic peptide.
  • a method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide sterically prevents the alpha-helical structure of the cytotoxin from forming.
  • the cytotoxic peptide can be modified to include negatively charged amino acids, thereby preventing the toxic pore conformation from forming.
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits could include the procytotoxins discussed in certain embodiments of the methods, as well as the pharmaceuticat excipeint and other reagents required to use the procytotoxins as intended.
  • the double-stranded MMP2/melittin template was amplified by PCR using primer Pl, (SEQ ID NO: 19) and primer P2 (SEQ ID NO: 20) which has the 5' end phosphorylated.
  • PCR was carried out under the following conditions: 94°C for 5 min, then 28 cycles of: 94°C for 30 seconds, 62.5 0 C for 30 seconds and 72°C for 30 seconds; then 72°C for 10 min.
  • the PCR product was then purified using QIAGEN kit (cat. No.: 28104).
  • the LAP cDNA fragment was amplified using PCR with plasmid phTGFB-2 as template (ATCC # 59954).
  • a LAP sense primer containing a HindIII site (SEQ ID NO: 21) and a LAP antisense primer containing an EcoRI site (SEQ ID NO: 22) were used in the PCR reaction.
  • PCR was carried out under the following conditions: 94°C for 5 min, then 25 cycles of: 94 0 C for 30 seconds, 75°C for 30 seconds and 72°C for 30 seconds; then 72 0 C for 10 min.
  • the PCR product was then purified using QIAGEN kit (cat. No.: 28104) and digested with HindIII and EcoRI.
  • the pCRMMP2/melittin plasmid was also digested with HindIII and EcoRI.
  • a mouse DHFR cDNA fragment was cut out from plasmid pS V2-DHFR (ATCC# 37146) using BstXl and Not!
  • the ⁇ IRES2-EGFP plasmid was also digested with BstXl and Notl (Clontech). 231.
  • the BstXl/Notl digested DHFR cDNA fragment and the BstXl/Notl digested pIRES2-EGFP plasmid were then purified.
  • the BstXl/Notl digested DHFR cDNA fragment and the BstXl /Notl digested pIRES2-EGFP plasmid were then incubated together under conditions suitable to allow insertion of the digested DHFR cDNA fragment to insert itself into the digested pIRES2-EGFP plasmid.
  • the plasmid was named as pIRES/DHFR. 232.
  • the LAP/mmp2/melittin fragment was cut out of pCRLAP/mmp2/melittin with HindIII and Xhol and inserted into the pIRES/DHFR plasmid cut with Smal.
  • the resulting mammalian expression vector was named as pLAP/mmp2/melittin/IRES/DHFR.
  • CHO/DHFR- (ATCC# 9096) cells were transfected with the pLAP /mmp2/melittin/IRES/DHFR plasmid and selected with G418 in medium containing
  • DMEM+1.5g/L sodium bicarbonate O.lmM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, 10% FBS, Ix PSN, and 1000 ug/ml G418.
  • the LAP-MMP2-Melittin construct was cloned into pIRES-DHFR vector.
  • This plasmid DNA was then transfected into CHO cells as described in Example 1. Forty- eight hours after transfection, cells were selected with G418 in medium containing DMEM+1.5g/L sodium bicarbonate, O.lmM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, 10% FBS, Ix PSN, and 1000 ug/ml G418. Cells were continuously cultured in G418 media containing 10% FBS. Once G418 selection was complete, the transfected CHO cells were adapted to media containing only 1% FBS. 237.
  • Transfected CHO cells were induced to produce the LAP-MMP2-Melittin fusion protein by increasing the concentration of MTX in culture media from 5OnM MTX to 500 nM MTX.
  • cell lysate was obtained and analyzed via SDS PAGE followed by western analysis with LAP antiserum (Figure 1).
  • Western analysis revealed a protein with the predicted molecular weight of the LAP fusion protein. This protein was the most intense at MTX concentrations of 250nm and 500nm. An intense band is also seen at the 5OnM MTX concentration, but there was also a mixture of other bands.
  • the next step was to see if the LAP fusion protein was secreted into the culture media. This was examined by collecting media from transfected cells in the presence of 50OnM MTX. Prior to media collection, cells were exposed to each of the following concentration of MTX; 50nm, 10OnM, 25OnM, and 50OnM, for 48 hours. Culture media was then collected from cells exposed to 500 nM MTX, cellular debris was pelleted, and the supernatant was stored at -20 degrees Celsius. In order to concentrate the amount of fusion protein in the culture media, the media was lyophilized. Lyophilized media was then resuspended in water in a volume approximately 1 OX less than the initial volume. LAP
  • MMP2 Melittin fusion protein was partially purified from the concentrated culture media by hLAP affinity chromatography using a batch procedure. The sample was then analyzed via native PAGE followed by western blotting with hLAP antiserum (Figure 3). This sample was compared to protein obtained from transfected cell lysate. The same bands were seen for both of these samples on the western.
  • LAP MMP2 Melittin was also purified from culture media via a different method. The sample was lyophilized as described above and then a size exclusion column on the Water's HPLC was used for purification. Figure 4 shows the partially purified protein.
  • Example 3 In Vitro Target Cell Killing
  • LAP MMP2 Melittin fusion protein can also be assessed.
  • DU- 145 prostate cancer cells which secrete MMP, 2 can be plated in a 48 well plate on day 0. On day 1 , these cells can be treated with the fusion protein for 3 hours, and then cell lysis will be determined via an LDH (lactate dehydrogenase) assay. The reaction can be visualized by an LDH assay.
  • LDH lactate dehydrogenase
  • purified LAP MMP2 Melittin fusion protein can be incubated overnight at 37 degrees Celsius in the presence or absence of active MMP2. The following day, the treated and untreated samples can be incubated with CHO cells. Cells can be treated for 1 hour and then an LDH assay can be done to determine if the cells have been lysed. Treatment of the fusion protein overnight with active MMP2 can cleave the protein at the MMP2 cutting site and release free melittin. When the MMP2 treated fusion protein is incubated with CHO cells, the cells show increased cell lysis as compared to cells incubated with untreated fusion protein.
  • LAP MMP2 Melittin fusion protein Functionality of the LAP MMP2 Melittin fusion protein can also be shown in vivo.
  • C57 female mice can be inoculated s.c. with approximately 0.8 million Bl 6 cells. 7 days after tumor inoculation, mice can be injected Lm. with 50ug of LAP MMP2 Melittin plasmid DNA. Mice can then be injected two additional times per week. Tumor size is reduced and longevity of the mice is increased.

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Abstract

Disclosed are procytotoxins that comprise a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide. Also disclosed are compositions and methods for selectively destroying a target cell. Also disclosed are compositions and methods for treating cancer in a patient. Also disclosed are compositions and methods for providing latency to a cytotoxin.

Description

LATENT PROCYTOTOXINS AND USES THEREOF
FIELD OF INVENTION
1. This invention relates to methods and compositions for selectively destroying a target cell. Specifically, the invention relates to methods for making and using a procytotoxin that can be converted to a cytotoxin in a target-cell specific manner, such that a target cell is destroyed by the activated cytotoxin.
BACKGROUND
2. Present methods for tumor treatment, especially cancer treatment, remain sub- optimal and often are accompanied by severe complications. In fact, virtually all of the known therapies have serious adverse side effects, most often caused by the lack of specificity and thoroughness in the destruction or removal of tumor or cancer cells.
3. For example, surgery, a common procedure for removing cancerous cells from a patient, often results in incomplete removal and disfigures the patient or interferes with normal bodily functions. Similarly, chemotherapy and radiation treatment often indiscriminately destroys normal cells, causing unwanted side-effects, while leaving many cancer cells unaffected. Chemotherapeutic agents, especially antimetabolites, while effective to varying degrees against cancer cells that are continuously undergoing or preparing for mitosis, are not effective against cancer cells that are in the resting (Go) stage. 4. Cancer treatment is most effective when cancer cells can be eliminated as completely as possible from the patient's body. To achieve this goal, continuous or consecutive dosages are administered to the patient. Because most available chemotherapeutic agents are also toxic to normal cells, the dose of cytotoxic drug is adjusted to the limits of tolerance to achieve the maximum destruction of malignant cells, and the interval between doses must be such that the rate of tumor re-growth does not exceed tumor killing. Accordingly, in order to achieve increased efficiency with reduced side effects, the chemotherapeutic agents should have high target-cell specificity and high target-cell toxicity or potency. In spite of considerable research into therapies for cancer, currently available treatment methods are ineffective in a significant percentage of cases. 5. Accordingly, there is a need for improved cancer treatment and more methods that are not dependent upon the cell cycle of the cancer cell. The present invention fulfills these needs and further provides other related advantages. SUMMARY
6. This invention relates to methods and compositions for selectively destroying a target cell. Specifically, the invention relates to methods for making and vising a procytotoxin (where the cytotoxin is maintained in an inactive form) that can be converted to a cytotoxin in a target-cell specific manner, such that a target cell is destroyed by the activated cytotoxin.
7. It therefore is an object of the present invention to overcome some or all of the aforementioned deficiencies in conventional therapies, especially with regard to treating cancer. To this end, compositions and methods for making a procytotoxin are described. A procytotoxin is provided which typically is made up of, for example, a cytotoxic agent bound to a latency associated peptide (LAP) via a peptide bond, and the peptide bond is susceptible to cleavage by a tumor specific protease. Thus, the procytotoxin can be described as V-Y-X, where V is a cytoxic agent, Y is a proteolytic cleavage site and Y is an LAP. The cytotoxic agent can be, for example a cytotoxic peptide. The peptide bond can be a proteolytic cleavage site. The procytotoxin of the present invention can also have a targeting molecule linked to the N- and/or C-terminus of the cytotoxic peptide.
8. In order to enhance its therapeutic usefulness, a cytotoxin is modified according to the invention to render it to a non-toxic form, known as a procytotoxin. A procytotoxin is a cytotoxic agent that can be rendered non-toxic by providing a protective layer around the cytotoxic agent, hereinafter referred to as "caging". The LAP can provide a protective "cage" around the pharmaceutically active agent thereby shielding it and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity. The procytotoxin can also be a cytotoxic peptide that is rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic. Specifically, the procytotoxin can comprise a cytotoxic peptide bound to a LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease. The LAP hinders the cytotoxic peptide from forming an active conformation. For example, when the procytotoxin contacts a target cell, a tumor specific protease, for example resident in the tumor cell membrane or extracellular matrix, cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane. Accordingly, a method for making a procytotoxin, comprising modifying a cytotoxic peptide to include a LAP, is described.
9. Also provided are pharmaceutical compositions that contain a disclosed procytotoxin and a pharmaceutically suitable excipient. 10. Further, methodology is provided for selectively destroying a target cell. The disclosed approaches typically entail contacting the target cell with a procytotoxin, which has a cytotoxic agent bound via a peptide bond to an LAP, wherein the peptide bond is susceptible to cleavage by a target specific protease. Target cells can be cells that are involved in the microvasculature surrounding cancer cells and cancer cells. 11. Further provided is a method of treating cancer in a patient. In general, the method comprises administering to a patient a therapeutically effective amount of a procytotoxin. In some embodiments, procytotoxins can be based on an amoebopore, melittin, or a cytolytic peptide derived therefrom.
12. Further methodology is provided for providing latency to a cytotoxin. In general, the method comprises covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin.
Figure Descriptions
13. Figure 1 shows the detection of hLAP-MMP2-Melittin in transfected CHO cells. 30 ug [search for ug/ul througout] of LAP CHO lystate (hLAP-MMP2-Melittin transfected) and CHO lysate (Not Transfected) was analyzed via SDS PAGE followed by western blotting with hLAP antiserum. 50ng of hLAP was used as a positive control.
14. Figure 2 shows methotrexate induction of LAP-MMP2-Melittin production in transfected CHO cells. CHO cells were transfected with LAP-MMP2-Melittin plasmid DNA in the presence of normal transfection media. Upon transfection cells were cultured in media containing 10% FBS and 5OnM MTX. The amount of FBS was gradually decreased in the media to 1 %. Cells were then induced to produce protein by increasing the amount of MTX in the media. Lysate was obtained from cells at different MTX concentrations and analyzed via SDS PAGE followed by western blotting with h LAP antiserum. Approximately 30 ug of lysate was analyzed for each MTX concentration and 50ng of hLAP (western positive control) was analyzed. 15. Figure 3 shows analysis of lysate and supernatant from hLAP-MMP2-Melittin transfected CHO cells under nondenaturing conditions. hLAP fusion protein (hLAP-MMP2- Melittin) present in culture media was concentrated prior to analysis via affinity chromatography. 30 ug of fusion protein lysate was analyzed and 50ng of hLAP was used as a positive control. All samples were analyzed via Native PAGE followed by western blotting with hLAP antiserum.
16. Figure 4 shows analysis of partially purified hLAP-MMP2-Melittin fusion protein. Purified hLAP-MMP2-Melittin Fusion protein was analyzed via SDS PAGE followed by western blotting with hLAP antiserum. The fusion protein was partially purified via size exclusion chromatography on the Waters HPLC system.
DETAILED DESCRIPTION
17. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, 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.
Definitions
18. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.
19. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment 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," it will be understood that the particular value forms another embodiment. 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 when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. 20. hi this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
21. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 22. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation. 23. "Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 24. Unless otherwise indicated by context, the term "contacting the target cell with a procytotoxin" means bringing the procytotoxin and target cell into close enough proximity with one another such that the a protease expressed by the target cell or resident in extraceullar matrix can cleave the proteolytic cleavage site of the procytotoxin, thus releasing the LAP from the cytotoxin and thereby allowing the cytotoxin to become active and exert its function on the target cell.
25. With regard to the terminology used herein, the artisan will recognize that a "standard" peptide bond is formed between the alpha carboxyl group of one amino acid with the alpha amino group of the next amino acid in the peptide chain and that peptide sequences are read from their amino-terminal end to their carboxyl-teraiinal end.
26. Unless otherwise indicated by context, the term "RGD" refers not only to the peptide sequence Arg-Gly-Asp, it refers genetically to the class of minimal or core peptide sequences that mediate specific interaction with integrins. Thus, an "RGD targeting sequence" encompasses the entire genus of integrin-binding domains.
27. hi addition, unless otherwise indicated by context, the term "NGR" refers not only to the peptide sequence Asn-Gly-Arg, it refers genetically to the class of minimal or core peptide sequences that mediate specific interaction with integrin signal sequences that can be found on cells of the vasculature. For example, a NGR targeting sequence can be used to bind certain integrins and can serve as a useful targeting molecule to endothelial cells and other cells of the neo-vasculature. Thus, an "NGR targeting sequence" encompasses the entire genus of integrin-binding domains.
28. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Compositions
29. 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. For example, if a particular procytotoxin is disclosed and discussed and a number of modifications that can be made to a number of molecules including the procytotoxin are discussed, specifically contemplated is each and every combination and permutation of procytotoxin and the modifications that are possible unless specifically indicated to the contrary. 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 combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group 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. Thus, if there 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.
30. Disclosed are methods and compositions for selectively destroying a target cell. Specifically, the invention relates to methods for making and using a procytotoxin that can be converted to a cytotoxin at the site of a tumor-cell, such that a tumor cell is destroyed by the activated cytotoxin.
31. It therefore is an object of the present invention to overcome some or all of the aforementioned deficiencies in conventional therapies, especially with regard to treating cancer. To this end, compositions and methods for making a procytotoxin are described.
32. A procytotoxin is provided which typically is made up of a cytotoxic agent bound to a latency associated peptide (LAP) via a peptide bond, and the peptide bond is susceptible to cleavage by a tumor specific protease. Thus, the procytotoxin can be described as V-Y-X, where V is a cytoxic peptided, Y is a proteolytic cleavage site and Y is an LAP. The cytotoxic agent can be a cytotoxic peptide. The peptide bond can be a proteolytic cleavage site. The procytotoxin of the present invention can also have a targeting molecule linked to the N- and/or C-terminus of the cytotoxic peptide.
33. Cytotoxic peptides can be cytolytic peptides. Cytotoxins can include, but are not limited to: type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter ococcus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, streptolysin, analogs of the pore-forming cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
34. For example, type I bacterial exotoxins can be used as a cytotoxin. In addition, a type I bacterial exotoxin can be fused with a tumor cell membrane or tumor microenviroment targeting domain.
35. Type II bacterial exotoxins can also be used as a cytotoxin. In addition, type III bacterial exotoxins can either retain its own trans-membrane domin or it can be fused with a different domain such as TAT. 36. Examples of procytotoxins have the following structures: (1) Gly-Ile-Gly-Ala-
Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln- GIn-[Y]-(X), and (2) Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp- Lys-Leu-Ile-Gln-Leu-He-Glu-Asp-Lys-[Y]-(X), wherein (X) is an LAP and [Y] is a proteolytic cleavage site that can be oriented in either direction. 37. The LAP, for example can be the precursor peptide of TGFβ-1 , 1.2, 2, 3, 4, or 5.
The LAP can also be the precursor peptide of human TGFβ-1, 2 or 3 (SEQ ID NOS: 23- 25). The LAP can also be the precursor peptide of chicken TGFβ-4 (SEQ ID NO: 26). The LAP can also be the precursor peptide of frog TGFβ-5 (SEQ ID NO: 27).
38. The proteolytic cleavage site can be a MMP cleavage site, a PSA cleavage site or a GGH cleavage site. For example, the MMP proteolytic cleavage site can be a MMP 1, 2,
3, 7, 8, 9, or a MT-I-MMP cleavage site. The MMP proteolytic cleavage site can also be any of the MMP proteolytic cleavage sites listed in SEQ ID NOS: 28-100.
39. ha addition, the proteolytic cleavage site can be a PSA cleavage site with the sequence described by Volkel et al. (Engineering of human coagulation factor X variants activated by prostate-specific antigen, Molecular Biotechnology, January 2005, vol. 29, no. 1, pp. 19-30(12))
40. The procytotoxin can further contain a targeting molecule. For example, the targeting molecule can interact, bind, attach, combine, join, connect to, or with, a target cell or tissue. More specifically, the targeting molecule can be a neovascular targeting sequence of an anti-fibronectin ED-B antibody. The targeting molecule can be an RGD or NGR targeting sequence as described below. Any molecule that can target a specific tissue can be used as the targeting molecule of the present procytotoxin. For example, the targeting molecule can be an antibody that interacts with human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/pmell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM, alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm's tumor antigen-1, telomerase, melanoma gangliosides, or a simple transmembrane sequence.
41. Procytotoxins can also be charge neutralized in addition to comprising steric inhibitors of the cytotoxin's activity. For example, provided is [Gly-He-Gly-Ala-Val-Leu-
Lys(R)-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys(R)-Arg-Lys(R)-Arg- GIn-GIn-GIy-AIa-IIe-GIy-GIn-PrO]-(X), wherein (X) is an LAP as described herein, which is bound to the peptide marked in brackets via a peptide bond, wherein the peptide marked in brackets can be oriented in either direction, wherein the peptide bond is susceptible to cleavage by a tumor specific protease and wherein R is independently selected from the group consisting of the unmodified ε-amino group of the adjacent lysine residue, [ε-γ]-Glu, [ε-γ]-Glu-[α-γ]-(Glu)1-3, [ε-α]-(Phe)1-3, [ε-α]-(Tyr)1-3, [ε-α]-(Trp)1-3, [ε-α]-(Lys)1-3 and [ε- α]-(Arg)1-3, wherein [ε-γ] represents a peptide bond between the epsilon amino group of lysine and the gamma carboxyl group of the adjacent glutamate, [α-γ] represents a peptide bond between the alpha amino group of the first glutamate and the gamma carboxyl group of the second glutamate, [ε-α] represents a peptide bond between the epsilon amino acid of lysine and the alpha carboxyl group of the indicated amino acid and the subscript indicates that additional numbers of the designated amino acid can be linked to the first via conventional peptide bonds. Procytotoxins
42. hi order to enhance its therapeutic usefulness, a cytotoxin is modified according to the invention to render it to a non-toxic form, known as a procytotoxin. hi one embodiment of the present invention, the procytotoxin is a cytotoxic peptide that can be rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic. The procytotoxin can, alternatively, be a cytotoxic peptide that can be rendered non-toxic by providing a protective layer or shell around the cytotoxic peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity. Specifically, the procytotoxin comprises a cytotoxic peptide bound to a LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease. The LAP hinders the cytotoxic peptide from forming an active conformation. For example, when the procytotoxin contacts a target cell, a tumor specific protease cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane. Accordingly, a method for making a procytotoxin, comprising modifying a cytotoxic peptide to include a LAP, is also described. 43. Numerous methods of modification are available, the applicability of which will depend on the structural characteristics of the cytotoxin. A LAP, for example, may be added to either the N-terminus and/or C-terminus of the cytotoxic peptide to hinder the cytotoxic peptide from forming a conformation which renders the peptide toxic, therefore the cytotoxic peptide is rendered non-toxic. A LAP can also be added to either the N- terminus and/or C-terminus of the cytotoxic peptide, such that the LAP forms a protective shell around the cytotoxic peptide peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity. The LAP can also sterically preventthe alpha-helical structure of the cytotoxin from forming. Additionally, the cytotoxic peptide can be modified to include negatively charged amino acids, thereby preventing the toxic pore conformation from forming.
44. The procytotoxin of the present invention comprises a latency associated peptide and a cytotoxic agent, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic agent. In some embodiments, the cytotoxic agent can be a cytotoxic peptide or a cytolytic peptide, as described above. In other embodiments, the cytotoxic agent can be a melittin, a melittin analog, or a melittin derivative.
45. For instance, the procytotoxin of the present invention comprises the following structure:: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser- Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-[Y]-(X) wherein (X) is a LAP and [Y] is a proteolytic cleavage site that can be oriented in either direction. The LAP can be selected from the group consisting of the precursor peptide of TGFβ-1, 2, 3, 4, or 5. The LAP can also be the precursor peptide of human TGFβ-1, 2 or 3 (SEQ ID NOS: 23-25). The LAP can also be the precursor peptide of chicken TGFβ-4 (SEQ ID NO: 26). The LAP can also be the precursor peptide of frog TGFβ-5 (SEQ ID NO: 27).
46. The proteolytic cleavage site can be a MMP cleavage site, a PSA cleavage site or a GGH cleavage site. For example, the MMP proteolytic cleavage site can be a MMPl, 2, 3, 7, 8, 9, or 10 cleavage site. In one embodiment, the MMP cleavage site is the MMP2 cleavage site. Cleavage by a target specific protease at [Y] may yield a melittin peptide with a few additional amino acids on the C-terminus which should not interfere with pore formation.
47. Also contemplated in the instant invention is a targeting molecule that adds an additional measure of selectivity. The targeting molecule may direct the procytotoxin in or around the cell, as well as act as a LAP, maintaining the cytolytic peptide in its inactive conformation. A targeting molecule of the instant invention can be selected from the group consisting of a signal sequence or an antibody. In some embodiments, the targeting molecule can be an RGD targeting sequence. 48. It is therefore also understood that in addition to or instead of steric determinants, charge determinants are also important. Where the positive charge of the pore forming peptide is involved in its cytolytic activity, eliminating/neutralizing the charge renders the peptide non-toxic. It is envisioned that even a partial neutralization of this positive charge will be effective. Accordingly, disrupting the alpha helical structure of the cytotoxin by sterically preventing the structure from forming in addition to eliminating/neutralizing the positive charge of the pore-forming peptide is also disclosed herein. Thus, even if no neutralization is accomplished, some of the modifications below will result in steric alterations which will inactivate the toxin to create the protoxin.
49. The procytotoxic peptides of this invention maybe chemically synthesized by standard solid phase procedures using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide. A combination of manual and automated (e.g., APPLIED BIOSYSTEM. 430A) solid phase techniques can be used to synthesize the novel peptides of this invention. For background on solid phase techniques, reference is made to Andreu et al, (1983) Proc. Natl. Acad. Sci USA 80: 6475-6479; Andreu et al., (1985) Biochemistry 24: 1683-1688; Fink et al. (June 1989) Int. J. Peptide Protein Res. 33: 412-421; Fink et al, (1989) J. Biol. Chem. 264: 6260-6267; each of which is incorporated herein by reference. 50. The in vivo stability of the procytotoxin of the invention can be improved by adding a D-amino acid to the N- or C-terminus, whichever does not have a γ-linked glutamic acid residue. Some in vivo instability might be advantageous because it could decrease the chance of possible adverse side effects that might arise once the procytotoxin is converted to cytotoxin. This procedure is particularly useful with products of the invention which are employed under conditions, parenteral or oral, where they will be subject to hydrolysis by naturally occurring enzymes before they perform their desired physiological function.
Cytotoxic Peptides 51. Many naturally occurring and synthetic cytotoxic peptides are known in the art.
Some are useful as therapeutic agents against pathogenic bacteria and other classes of microorganisms; they may be isolated from insects, frogs and other animals. Specific examples include alamethicins, attacins, bactenecins, cecropins (see Table 1 the amino acid sequences of cecropins A, B and C), CytA and CytB of Bacillus thurigiensis, defensins, enterocin L50 (pediocin L50), lantibiotics, magainins, PGLa, protegrins, sapecin, and sarcotoxin.
Cytolytic Peptides
52. In some embodiments, the cytotoxic peptide is a cytolytic peptide. Cytolytic peptides, also known as pore-forming or channel-forming peptides, typically disrupt cell membranes, causing cell lysis and death upon contact. Many naturally occurring cytolytic peptides from microorganisms, insects and higher animals are generally known. They often are called hemolysins because they lyse red blood cells as well as other eukaryotic cells. These toxins include type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I and other cytolysins of sea anemone, aerolysin, amatoxins, amoebapores, amoebapore homologs from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter ococcus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxins, enterotoxin oϊAeromonas hydwphila, esculentin, granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin and neokyotorphin fragments (1-4), NK-lysin, paradaxins, perforin (especially its amino terminus), perfringolysin O (PFO or theta-toxin) of Clostridium perfringens, phallolysins, phallotoxins, streptolysins. Some hemolysins like melittin are also known to kill bacteria. Other cytolytic peptides include D,L-α-amino acid cyclic peptides. Fernandez-Lopez et al., Nature, 412:452-455 (2001).
53. For example, type I bacterial exotoxins can be used as a cytotoxin. In addition, a type I bacterial exotoxin can be fused with a tumor cell membrane or tumor microenviroment targeting domain.
54. Type II bacterial exotoxins can also be used as a cytotoxin. In addition, type III bacterial exotoxins can either retain its own trans-membrane domin or it can be fused with a different domain such as TAT. Pore-Forming Cytolytic Peptides
55. The cytolytic peptide can be a pore-forming or channel-forming cytolytic peptide. Many cytolytic peptides are pore-forming toxins, belonging to a group of cytotoxins that associate with cell membranes, either nonspecifically or to specific receptors, and form transmembrane pores of discrete size. Most toxic pore-forming peptides employ common features for their cell lysis activity. For example, a great number of these toxins lyse cells through a "barrel-stave" mechanism, in which monomers of the toxin bind to and insert into the target membrane and then aggregate like barrel staves surrounding a central, water filled pore. This pore causes rapid and irreversible electrolyte imbalance of the target cell leading to its destruction. 56. Most pore-forming peptides that act on both mammalian and bacterial cells require an amphipathic alpha-helical structure and a net positive charge for their cytolytic activity. A strong electrostatic interaction between the cationic portion of the peptide and the lipid headgroups weakens the membrane, facilitating insertion of the hydrophobic alpha- helical peptides. Accordingly, a cytotoxic peptide according to the invention can be a cytolytic, linear α-helical peptide. Generally these cytotoxic peptides, in their native form, will have a net positive charge, which contributes to their cytolytic activity.
Melittin
57. According to an embodiment of the invention, the cytolytic peptide can be melittin or an analog or derivative thereof. Melittin is isolated from bee venom and is a 26 amino acid amphiphilic alpha-helix (Blondelle et al., (1991) Biochemistry 30: 4671-4678; Dempsey et al., (1991) FEBS Lett. 281: 240-244.) The amino acid sequence of melittin is shown in Table 1 and SEQ ID NO: 6. Residues 1-20 are predominantly hydrophobic and residues 21 to 26 are hydrophilic and basic. Melittin has antibiotic activity, but in mammals it is lytic for leukocytes, red blood cells and a wide variety of other cells. Compounds similar to melittin, which are also within the scope of the invention, include bombolitin from bumblebee venom (17 amino acid amphiphilic alpha-helix), mastoparan from wasp venom (14 amino acid amphiphilic alpha-helix) and crabrolin from hornet venom (13 amino acid amphiphilic alpha-helix) (Argiolas A. and Pisano J. J., 1985, J. Biol. Chem. 260, 1437- 1444.).
TABLE 1
Amino Acid Sequence of Selected Cytolytic Peptides
Cecropin A {Antheria pernyi)
NH2-Lys-Tφ-Lys-Leu-Phe-Lys-Lys-ne-Glu-Lys-Val-Gly-Gln-Asn- He- Arg-Asp-Gly-He-Ile-Lys-Ala-Gly- Pro-Ala- Val-Ala- VaI- Val-Gly- Gln-Ala-Thr-Gln-Ile-Ala-Lys-COOH
Cecropin B {Antheria pernyi)
NHrLys-Trp-Lys-Ile-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Arg-Asn-Ile- Arg- Asn-Gly-Ile-Ile-Lys-Ala-Gly-Pro-Ala- Val-Ala- Val-Leu-Gly-Glu-Ala-
Lys- Ala-Leu-COOH
Cecropin D {Antheria pernyi)
NH2-Trp- Asn-Pro-Phe-Lys-Glu-Leu-Glu-Lys-Val-Gly-Gln-Arg- VaI- Arg-
Asp-Ala-Val-Ile-Ser-Ala-Gly-Pro-Ala-Val-Ala-Thr-Val-Ala-Gln-Ala- Thr- Ala-Leu-Ala-Lys-COOH Melittin (Apis mellifera)
NH2-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro- AIa-
Leu-Ile-Ser-Trp-ne-Lys-Arg-Lys-Arg-Gln-Gln-COOH
Amoebapore Helix 3 (Entamoeba histolytica)
N^-Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly- Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu- Asp-Lys-COOH
Amoebapore
58. Another pore-forming peptide, according to the instant invention, can be amoebapore, a 77-residue pore-forming peptide from the amoebae Entamoeba histolytica (Young et al, (1982) J. Exp. Med. 156: 1677; Lynch et al, (1982) EMBO J 7: 801; Young & Cohn, (1985) J. Cell Biol. 29: 299; Rosenberg etal, (1989) Molec. Biochem. Parasit. 33: 237; Jansson et al., (1994) Science 263: 1440). It has four alpha helices, from amino acids approximately 1-21, 25-36, 40-63 and 67-77, conventionally called helices 1, 2, 3, and 4, respectively. 59. The amoebapore peptide is stabilized by three disulfide bonds and contains four mostly amphipathic alpha-helical structures. The third amphipathic helical structure (helix 3) retains the cytolytic activity similar to the wild type peptide. A synthetic peptide based on the sequence of its third amphipathic alpha helix has recently been shown to have cytolytic activity for nucleated cells at high concentrations (10-100 μM) (Leippe et ah, (1994) Proc. Natl. Acad. Sci. USA 91 : 2602). Accordingly, in one embodiment the cytotoxin is an amoebapore derivative listed in Table 1 : NKb-Gly-Phe-Ile-Ala-Thr-Leu-Cys- Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-COOH.
Modifications and Derivatives 60. It is readily recognized that the above described cytolytic peptides may be ' modified or derivatized to produce analogs and derivatives which retain, or even exhibit enhanced, cytolytic activities. For example, Andra et al. disclose that shortened amoebapore analogs have enhanced antibacterial and cytolytic activities (FEBS Letters, 385 (1996), pp. 96-100, incorporated herein by reference in its entirety). 61. In designing such analogs or derivatives, the artisan will be informed by the foregoing discussion relating to the amphipathic alpha-helical structure and net positive charge that are implicated in the cytolytic activity. Thus, a skilled artisan is able to design cytotoxin analogs, i.e., non-native forms never before known in nature, based on the observed homologies and known structure and properties of the native protein, to be used as a cytolytic peptide in accordance with the instant invention.
62. Modification and derivatization according to the instant invention include, but are not limited to, substitutions, additions or deletions that provide for functionally equivalent molecules. (Function may be assessed in accord with the working examples presented below.) Analogs and derivatives may also be made via modifications of side chains of amino acid residues of the cytotoxic peptides, for example, the analogs or derivatives can have enhanced or increased functional activity relative to the native protein or polypeptide.
63. Dimerization, truncation, diasteroisomers (D-amino acid-incorporated analogs) (Shai et al, (1996) J. Biol. Chem. 271-7305-7308), and combinations thereof may also be employed for production of alternate therapeutic forms, derivatives and analogs of cytotoxin peptides. For example, Shai et al., (1996) J. Biol. Chem. 271-7305-7308, discloses the effect of sequence and structural variations on the cytolytic activity of melittin, and is hereby incorporated by reference in its entirety. Sequence similarities
64. Homologs of the present peptides are provided. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not. 65. hi general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
66. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison maybe conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
67. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger zt ύ. Methods En∑ymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
68. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
Nucleic acids 69. There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, melittin as well as any other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. Nucleotides and related molecules
70. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'- AMP (3 '-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
71. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. 72. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
73. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556),
74. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
75. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
Sequences
76. There are a variety of sequences related to, for example, melittin as well as any other protein disclosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.
77. A variety of sequences are provided herein and these and others can be found in Genbank, at www.pubmed. gov. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art. Peptides and Proteins
78. There are numerous variants, modifications or derivatives of the disclosed procytotoxin proteins that disclosed and herein contemplated. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions can be made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions. TABLE 2: Amino Acid Abbreviations
79. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.
80. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
81. Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
82. Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o- amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl. 83. It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. For example, SEQ ID NO: 6 sets forth a particular sequence of melittin and SEQ ID NO: 14 sets forth a particular sequence of a amoebapore protein. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to the stated sequence. In one embodiment, the variant retains the sole tryptophan residue at position 19 in the disclosed melittin sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
84. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. 85. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. 86. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.
87. As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 6 is set forth in SEQ ID NO: 5. Another nucleic acid sequence that encodes the same protein sequence set forth in SEQ ID NO: 14 is set forth in SEQ ID NO: 13. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular procytotoxin from which that protein arises is also known and herein disclosed and described.
88. It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 2 and Table 3. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs). 89. Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH-, -CH2S-, -CH2-CH2 --, -CH=CH- (cis and trans), -COCH2 --, ~ CH(OH)CH2-, and -CHH2SO- (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (-CH2NH-, CH2CH2-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH H2-S); Hann J. Chem. Soc Perkin Trans. 1307-314 (1982) (--CH-CH-, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH2-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH2-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-- CH(OH)CH2-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (-C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982) (-CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH2NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like. 90. Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
91. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference) .
Latency Associated Peptide (LAP) Function/Mechanism
92. A latency associated peptide (LAP) according to the present invention is an object that, when attached to a lytic peptide, inactivates the cytotoxin by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic.
93. The LAP can vary in size. So long as the amino acids of the LAP cage, charge neutralize and/or create steric restriction of lytic peptide membrane insertion and organization into a pore, the peptide is a suitable LAP. 94. For example, the LAP can be the precursor domain of TGFβ or a sequence which is substantially identical thereto. The LAP can also be a phage or phage particle. 95. TGFβ can be synthesized as a dimeric latent cytokine composed of an amino acid terminal latency associated protein (TGFβ-LAP) and the active TGFβ cytokine at its COOH terminal end (Roberts and Sporn, Peptide Growth Factors and their Receptors: Sporn, M B and Roberts, A B, Springer- Verlag, 419-472 (1996); Roth-Eicchorn et al., Hepatology, 28 1588-1596 (1998)). The precursor peptide can contain a signal peptide (residues 1-29) necessary for protein secretion and guiding the molecule through the Golgi apparatus to become processed by proteolytic cleavage and glycosylation. The TGFβ-LAP domain can be separated from TGFβ by proteolytic cleavage at arginines (277-278). Mature TGFβ begins at alanine 279. The TGFβ-LAP, in addition to preserving TGFβ in a latent form, contains important residues necessary for the interaction with other molecules. Mutations in the TGFβ-LAP domain have recently been associated with the autosomal dominant Camurati-Engelmann disease (Janssens et al., Nature Genetics, 26, 273:275 (2000). Cysteines 224 and 226 are important in the intermolecular disulphide bond between two TGFβ-LAP proteins. Their mutation to serine renders the molecule "active" (Sanderson et al., Proc. Natl. Acad. Sci. USA, 92, 2572-2576 (1995); Brunner et al., MoI. Endocrinol. 6, 1691-1700 (1992); Brunner et al., J. Biol. Chem, 264, 13660-13664 (1989)). The RGD motif (245-247) facilitates the interaction with integrins (Munger et al., MoI, Biol, of the Cell, 9, 2627-2638 (1998; Derynck R, TIBS, 19, 548-553 (1994)). The nucleic acid encoding TGFβ is described in U.S. Pat. No. 5,801,231 and is herein incorporated by reference for its disclosure of the nucleic acid sequence encoding TGFβ-LAP.
96. In most cell types studied, including those of mesenchymal, epithelial and endothelial origin, TGFβ is secreted in a latent form consisting of TGFβ subunit and TGFβ- LAP propeptide dimers, covalently linked to latent TGFβ-binding proteins (LTBPs). LTBPs are also needed for the secretion and folding of TGFβ (Miyazano et al., EMBO J. 10, 1091-1101 (1991); Miyazano et al., J. Biol. Chem. 267, 5668-5675 (1992); Eklov et al., Cancer Res. 53, 3193-3197 (1993)). Cysteine 33 is important for the disulphide bridge with the third 8 cysteine-rich repeat of latent TGFβ binding protein (LTBP) (Saharinen et al., The EMBO Journal, 15, 245-253 (1996). Modification of LTBP by enzymes such as thrombospondin (Schultz et al., The Journal of Biological Chemistry, 269, 26783-26788 (1994); Crawford et al., Cell, 93, 1159-1170 (1998)), transglutaminase (Nunes et al., J. Cell, Biol. 136, 1151-1163 (1997); Kojima et al., The Journal of Cell Biology, 121, 439-448 (1993)) MMP9, and MMP2 (Yu and Stamenkovic, Genes and Dev, 14, 163-176 (2000)) could release the active portion of TGFβ from the latent complex.
97. The LAP of the present invention may comprise the precursor domain of TGFβ, for example, the precursor peptide of TGFβ -1, 2 or 3 (from human) (Derynck et al., Nature, 316, 701-705 (1985); De Martin et al., EMBO J. 6 3673-3677 (1987); Hanks et al., Proc. Natl. Acad. Sci. 85, 79-82 (1988); Derynck et al., EMBO J. 7, 3737-3743 (1988); Ten Dyke et al., Proc. Natl. Acad. Sci. USA, 85, 4715-4719 (1988)), TGFβ-4 (from chicken) (Jakowlew et al., MoI. Endocrinol. 2, 1186-1195 (1988)), or TGFβ-5 (from xenopus) (Kondaiah et al., J. Biol. Chem. 265, 1089-1093 (1990)). The term "precursor domain" is defined as a sequence encoding a precursor peptide and does not include the sequence encoding the active TGFβ protein
98. In some embodiments, the amino acid sequence of the LAP can have at least 50% identity, using the default parameters of the BLAST computer program (Atschul et al., J. MoI. Biol. 215, 403-410 (1990) provided by HGMP (Human Genome Mapping Project), at the amino acid level, to the precursor domain of TGFβ 1, 2, 3, 4 or 5 (Roberts and Sporn, Peptide Growth Factors and their Receptors: Sporn, M B and Roberts, A B, Springer- Verlag, Chapter 8, 422 (1996)). hi other embodiments, the LAP can have at least 60%, 70%, 80%, 90%, or 95% identity, at the nucleic acid or amino acid level, to the LAP of TGFβ 1, 2, 3, 4 or 5. In another embodiment the LAP can have up to or above 99% identity, at the nucleic acid or amino acid level, to the LAP of TGFβ 1, 2, 3, 4 or 5.
99. The LAP can be seleted from the LAPs of SEQ ID NOS: 23-27.
100. The LAP may contain at least two, for example at least 4, 6, 8, 10 or 20 cysteine residues for the formation of disulphide bonds.
101. The LAP may provide a protective "cage" around the pharmaceutically active agent thereby shielding it and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity.
102. The LAP can comprise the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 1 or nucleotides 103-933 of SEQ ID NO: 3 or a sequence which has at least 50%, 60%, 70%, 80%. 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
103. The LAP can comprise the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 9 or nucleotides 97-928 of SEQ ID NO: 11 or a sequence which has at least 50%, 60%, 70%, 80%. 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
Location 104. The LAP can be added to the N- or C-terminus, or both the N- and C- terminus, of the cytotoxic peptide. For example, the cytotoxic peptide can be a cytolytic peptide, and the LAP can be added to the C-terminus of a cytolytic peptide. Furthermore, the LAP may additionally comprise a targeting molecule, as discussed herein.
105. hi addition, a charge neutralization component can be added to the procytotoxin where the procytotoxins further are charge neutralized, in addition to steric determinants.
106. For example, provided is a peptide comprising [Gly-Ile-Gly-Ala-Val-Leu- LysC^-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-LysC^-Arg-LysC^-Arg- GIn-GIn-GIy-AIa-IIe-GIy-GIn-PrO]-(X), wherein (X) is the LAP as described herein, which is bound to the peptide marked in brackets via a peptide bond, wherein the peptide marked in brackets can be oriented in either direction, wherein the peptide bond is susceptible to cleavage by a tumor specific protease and wherein R is independently selected from the group consisting of the unmodified ε-amino group of the adjacent lysine residue, [ε-γ]-Glu, [ε-γ]-Glu-[α-γ]-(Glu)1-3, [ε-α]-(Phe)1-3, [ε-α]-(Tyr)1-3, [ε-α]-(Trp)1-3, [ε-α]-(Lys)1-3 and [ε- α]-(Arg)1-3, wherein [ε-γ] represents a peptide bond between the epsilon amino group of lysine and the gamma carboxyl group of the adjacent glutamate, [α-γ] represents a peptide bond between the alpha amino group of the first glutamate and the gamma carboxyl group of the second glutamate, [ε-α] represents a peptide bond between the epsilon amino acid of lysine and the alpha carboxyl group of the indicated amino acid and the subscript indicates that additional numbers of the designated amino acid can be linked to the first via conventional peptide bonds.
Target Specific Protease Function/Mechanism/Location
107. A target specific protease as described herein is an enzyme associated with a target cell that cleaves a peptide bond of proteins. A target cell, as described herein, can be any cell whose lysis is desired. For example, a target cell could be a cancer cell, a bacterial cell, a neuronal cell, a bone marrow cell, a prostate cell, or an endothelial cell of the tumor neo-vasculature.
108. The targeting specific protease of the present invention may be typically expressed in tumors, but need not be exclusively expressed in tumors. Accordingly, the targeting specific protease may be preferentially expressed in tumor cells when compared to normal cells. Additionally, the targeting specific protease may be a protease expressed by a non-tumorigenic target cell, such as a bacterial cell, but is nevertheless preferentially expressed by a target cell when compared to a non-target cell. Accordingly, the procytotoxin as described herein is additionally useful as an antimicrobial or antibacterial agent. The procytotoxin as described herein is also additionally useful for lysing any target cell of interst, so long as the targeting specific protease is present in the target cell.
Matrix Metalloproteinase (MMP)
109. In some embodiments, the targeting specific protease can be a matrix metalloprotease (MMP). For example, the targeting specific protease can be MMP2. MMPs are associated with angiogenesis, as well as neo-vessel formation. Therefore, targeting the vasculature which supplies the tumors, as well as newly formed vessels, can serve to eliminate blood flow to tumors and can also prevent tumors from forming.
110. For example, cytolytic peptides linked to an LAP by a sequence of amino acids recognized by MMP can be selectively released from the LAP in the presence of a targeting specific protease such as MMP. The cytotoxic peptide is then free to form its active conformation and lyse target cells.
111. Additional targeting specific proteases suitable for the present invention include those described for PSA in Coombs et al, Chem Biol, 5(9):475-88 (1988), PSMA, and gamma-glutamyl hydrolase (GGH). Prostate Specific Antigen (PSA)
112. Additional reactive peptide substrates suitable for the present invention include those described for the prostate-specific antigen (PSA) in Coombs et ah, Chem Biol, 5(9):475-88 (1988). PSA is the product of the KLK3 gene, and is expressed by the prostate and other tissues. It is expressed by healthy cells but abnormally high levels of PSA in the blood are found in men prostate disorders including prostate cancer and in women with breast cancer. PSA is an important tumor marker used in the diagnosis and monitoring of both prostate and breast cancer. 113. PSA is a 33 -kd protein consisting of a single-chain glycoprotein of 237 amino acid residues, 4 carbohydrate side chains, and multiple disulfide bonds. PSA is homologous with the proteases of the kallikrein family. PSA may be referred to as human glandular kallikrein-3 (hK-3) to distinguish PSA from human glandular kallikrein-2 (hK-2), another prostate cancer marker with which it shares 80% homology. Human glandular kallikrein- 1 (hK-1) is a kallikrein that is found primarily in pancreas and renal tissue but shows 73% and 84% homology with PSA. The complete gene encoding PSA has been sequenced and localized to chromosome 19. PSA can be found in prostate epithelial cells and in the seminal fluid. 114. Serial analysis of human glandular kallikrein-3 gene expression and EST expression profiles has also shown upregulation of the human glandular kallikrein-3 gene in female genital (ovarian and uterine) and gastrointestinal (gastric, colon, esophageal and pancreatic) cancers. Significant downregulation was observed in breast cancers and brain tumors, in relation to their normal counterparts. (Diamandis et al., The Urologic Clinics of North America: Prostate-Specific Antigen: The Best Prostatic Tumor Marker (1997)). Currently, PSA is considered to be the most valuable tumor marker due to its tissue specificity and it is used widely for prostate cancer screening, diagnosis, and management.
115. The 5'-untranslated region of the gene encoding PSA contains regulatory elements, two of which are androgen response elements (ARE I and ARE II), and the other is a strong enhancer (Schuur et al., J Biol Chem (1996), Cleutjens et al., MoI Endocrinol (1997)). PSA gene transcription in the prostate is known to be regulated by androgens through the action of the AR (Riegman et al., MoI Endocrinol (1991); Schuur et al., J Biol Chem (1996); Cleutjens et al., MoI Endocrinol (1997); and Young et al., Cancer Res (1991)). hi seminal plasma, in which PSAis present at very high amounts (~l-2 g/liter), it appears that the role of PSA is proteolytic cleavage of the sperm motility inhibitor semenogelin, resulting in semen liquefaction post ejaculation (Malm J et al., Scand J Clin Lab Invest Suppl (1995) and McCormack et al., Urology (1995)). However, other substrates for PSA have been proposed including insulin-like growth factor binding protein 3 (IGFBP- 3), protein C inhibitor, transforming growth factor-β (TGF-β), PTH-related peptide (Cramer et al., J Urol (1996)), and an unknown precursor protein that releases a putative vasoactive peptide (Fichtner et al., J Urol (1996)). In male serum, PSA is present as a complex with Ot1- antichymotrypsin (PSA-ACT), α2-macroglobulin (PSA- A2M), and as free PSA (Stenman et al., Cancer Res (1991) and Christensson et al., Eur J Biochem (1990)). Prostate Specific Membrane Antigen (PSMA)
116. Another targeting specific protease can be a type II transmembrane protein such as the prostate specific membrane antigen (PSMA). It has been previously been discovered that prostate cancer cells over-express a type II transmembrane protein, the prostate specific membrane antigen. PSMA has an intracellular epitope that is immunoreactive toward the 7El 1C5 immunoglobulin G monoclonal antibody (Horosczewicz et al. (1983), LNCAP model of human prostatic carcinoma, Cancer Res. A2>: 1809-1818). hi prostate cancer patients, PSMA is highly expressed on malignant prostate epithelia, but only marginally on normal prostate glands, and to a lesser degree on benign prostatic hypertrophic epithelia. (Pinto et al. (1999) Prostate specific membrane antigen, a unique glutamate carboxypeptidase: a review of recent findings, The Prostate J. 1 : 15-26; Wright et al. (1995) Expression of Prostate Specific Membrane Antigen (PSMA) in normal benign and malignant prostate tissues, Urol. Oncol. 1 : 18-28; Lopes et ah, (1990) Immunohistochemical and pharmacokinetic characterization of the site-specific immunoconjugate CYT-356 derived from antiprostate monoclonal antibody 7El 1-C5, Cancer Res. 50: 623-6428; Troyer et al., Detection and characterization of the prostate- specific membrane antigen (PSMA) in tissue extracts and body fluids, Int. J. Cancer 62: 552-558).
117. The proteolytic domain of PSMA is located on the outside of the cell surface. Upon reaching the target cell, the procytotoxins of the invention, without having to be internalized by the target cell, will have their γ-glutamate residue(s) cleaved and removed. The procytotoxins thus can be activated precisely at the desired site of action, the target cell, as long as a similar mechanism is present to remove the LAP. This direct effect on the target cell increases the effectiveness of the prostate cancer cell killing. The activated cytolytic peptide immediately inserts into the target cell, leaving it almost no chance of acting upon adjacent, non-target cells. In fact, it is possible that one end of the peptide is already inserted into the membrane before it is activated by PSMA. Furthermore, as discussed above, the activated toxin is neutralized by the membrane therefore after the lysis of the target cell, the cytolytic peptides remain adsorbed in the membrane debris of the target cell and do not leak and harm non-target cells.
Gamma-Glutamyl Hydrolase (GGH)
118. Gamma-glutamyl hydrolase (GGH) is an intra-cellular enzyme. In certain types of cancers, GGH can secret into the tumor microenviroment.
119. GGH (EC 3.4.19.9) is a central enzyme in the metabolism of folyl and antifolyl poly-gamma-glutamates (McGuire, J. J., and Coward, J. K. (1984) in Folates and Pterins (Blakely, R. L. , and Benkovic, S. L., eds), Vol. 1 , pp. 135-190, John Wiley & Sons, Inc., NY). Folate is required as a cofactor by several enzymes in the de novo biosynthesis of DNA precursors and of several amino acids and is essential for normal cell growth and replication. Antifolates such as methotrexate (MTX) have been the traditional treatment for many cancers over almost four decades (Chabner et al., J. Clin. Invest. 76, 907-912). When folates or antifolates are transported into the cell, they are converted to folylpoly-gamma- glutamates or antifolylpoly-gamma-glutamates through the sequential addition of glutamates by folylpolyglutamate synthetase (FPGS) (EC 6.3.2.17). These poly-gamma-glutamates are retained intracellularlyand are generally better substrates or inhibitors than the corresponding monoglutamates for most of the folate-dependent enzymes (Galivan, J. (198O) Mo/. Pharmacol. 17, 105-110; Schirch, V., and Strong, W. B. (1989) Arch. Biochem. Biophys. 269, 371-380; Rhee, M. S., Lindau-Shepard, B., Chave, K. J., Galivan, J., and Ryan, T. J. (1998) MoI. Pharmacol. 53, 1040-1046).
120. GH catalyzes the removal of the gamma-linked polyglutamate chain, converting its substrates to folyl- or antifolyl-mono-gamma-glutamates, which are less well retained, and thus resulting in a reduction in the overall effectiveness of folates and antifolates. The roles of FPGS and GH activities in the regulation of the extent of polyglutamylation of folates or antifolate drugs are well demonstrated. Increased GH activity (Rhee, M. S., Wang, Y., Nair, M. G., and Galivan, J. (1993) Cancer Res. 53, 2227-2230; Yao, R., Rhee, M. S., and Galivan, J. (1995) MoI. Pharmacol. 48, 505-511) or decreased FPGS activity (Pizzorno et al., Cancer Res. 55, 566-573; Rumberger et al., Cancer Commun. 2, 305-310; McCloskey et al., J. Biol. Chem. 266, 6181-6187 ) have been associated with in vitro resistance to antifolates such as MTX, lO-propargyl-5,8- dideazafolate, and 5,10-dideazatetrahydrofolicacid. 121. In vivo, it has been reported that an increased GH activity may account for the inherent resistance to MTX in acute myelogenous leukemia (Rots et al., Blood 93, 1677- 1683). Recently, the ratio of FPGS/GH activities has been used as an indicator of MTX polyglutamylation in acute lymphocytic leukemia (Longo et al., Oncol. Res. 9, 259-263). Proteolytic Cleavage Site
122. The proteolytic cleavage site may comprise any protease specific cleavage site. The proteolytic cleavage site can include, but is not limited to, a matrix metalloproteinase (MMP) cleavage site, a PSA cleavage site, a PSMA cleavage site, or a GGH cleavage site. (Zhang et al., J. MoI. Biol. 289, 1239-1251 (1999); Voth et al., Molecular and Biochemical Parasitology, 93, 31-41 (1998); Yoshioka et al., Folia
Pharmacologica Japonica, 110, 347-355 (1997); Tort et al., Advances in Parasitology, 43, 161-266 (1999); McKerrow, International Journal for Parasitology, 29, 833-837 (1999); Young et al., International Journal for Parasitology, 29, 861-867 (1999); Coombs and Mottram, Parasitology, 114, 61-80 (1997)). 123. The MMP cleavage site may comprise any amino acid sequence which is cleavable by a MMP. The amino acid sequence of the MMP cleavage site may be encoded by nucleotides 832-852 of the procytotoxin shown in SEQ U) NO: 1 (melittin based) or nucleotides 832-852 of the procytotoxin shown in SEQ ID NO: 9 (amoebopore based) or a sequence of nucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto. For example, the nucleic acid sequence encoding the MMP cleavage site can comprise the minimum number of residues required for recognition and cleavage by MMP.
124. A MMP cleavage site may comprise a number of amino acid residues recognizable by MMP. Moreover, the amino acids of the MMP site may be linked by one or more peptide bonds which are cleavable, proteolytically, by MMP. MMPs which may cleave the MMP site include, but are not limited to, MMPl, MMP2, MMP3, MMP7, MMP8, MMP9, MMPlO or MTl-MMP (Yu and Stamenkovic, Genes and Dev. 14, 163- 176 (2000); Nagase and Fields, Biopolymers, 40, 399-416 (1996); Massova et al., J. MoI. Model. 3, 17-30 (1997); reviewed in Vu and Werb, Genes and Dev. 14, 2123-2133 (2000)). The sequences of the protein cleavage sites of MMPl, MMP2, MMP3, MMP7, MMP8, MMP9 and MMPlO are shown in SEQ ID NOS: 28-100. 125. In one embodiment, the proteolytic cleavage site of the present invention is cleaved at sites of tumor presence and tumor neo-vascular formation. For example, the proteolytic cleavage site of the present invention is a MMP cleavage site e.g any one or more of MMPl, MMP2, MMP3, MMP7, MMP8, MMP9 or MMPlO as shown in SEQ ID NOS: 28-100. In a specific embodiment, the cleavage site is a MMP2 cleavage site.
126. For example, cytotoxic peptides of the instant invention linked to an LAP by a sequence of amino acids recognized by MMP can be selectively released from the LAP in the presence of a tumor specific protease such as MMP. The cytotoxic peptide is then free to form its active conformation and destroy the target cells. Targeting Molecule
127. Selective delivery of therapeutic agents to cancer cells in a living body is another area of research where targeting of cancer specific biomarkers is intensively studied. (E. Mastrobattista, G. A. Koning, and G. Storm, "Immonoliposomes for the Targeted Delivery of Antitumor Drugs," Adv Drug Delivery Reviews 1999, 40: 103-27; J. Sudimack and R. J. Lee, "Targeted Drug Delivery Via Folate Receptor," Adv Drug Delivery Reviews 2000, 41 : 147-62; S. P. Vyas and V. Sihorkar, "Endogenous Carriers and Ligands in Non- Immunogenic Site-Specific Drug Delivery," Adv Drug Delivery Reviews 2000, 43: 101- 64.). Immunoliposome-mediated targeting using monoclonal antibodies to folate receptor, (E. Mastrobattista, G. A. Koning, and G. Storm, "Immonoliposomes for the Targeted Delivery of Antitumor Drugs," Adv Drug Delivery Reviews 1999, 40: 103-27; J. Sudimack and R. J. Lee, "Targeted Drug Delivery Via Folate Receptor," Adv Drug Delivery Reviews 2000, 41: 147-62) CA- 125, (E. Mastrobattista, G. A. Koning, and G. Storm, "Immonoliposomes for the Targeted Delivery of Antitumor Drugs," Adv Drug Delivery Reviews 1999, 40: 103-27) and HER2/neu antigen (D. B. Kirpotin, J. W. Park, K. Hong, S. Zalipsky, W. L. Li, P. Carter, C. C. Benz, and D. Papahadjopoulos, "Sterically Stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro," Biochemistry 1997, 36: 66-75) have been described.
128. . The targeting molecule of the instant invention is an optional feature that presents an extra measure of selectivity. The targeting molecule directs the procytotoxin to the target cell, where the procytotoxin is rendered toxic and selectively lyses its target. Additionally, the targeting molecule may act as an LAP of the cytolytic peptide. In this regard, the targeting molecule may charge neutralize or sterically inhibit a cytolytic peptide from pore formation. The targeting molecule may be added to the N- or C-terminus, or both.
129. In some embodiments, the targeting molecule is an antibody. For example, the antibody can be an anti-fibronectin ED-B antibody, thereby directing the procytotoxin to the extracellular matrix associated with neo-vessel formation.
130. Targeting molecules that target the neo-vasculature can also be added to the LAP. Molecules that target the neo-vasculature can easily be identified by screening phage display libraries. Any such peptide would be a suitable targeting molecule of the present invention. 131. Targeting molecules which bind specifically to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides bear the signal sequence based on Arg-Gly-Asp (RGD). Accordingly, sequences that bind certain integrins can serve as useful targeting molecules to endothelial cells and other cells of the neo-vasculature. For example, the targeting molecule can be an RGD targeting sequence. 132. Non-structural spacers may be a feature of the targeting molecule. Such spacers typically comprise glycine and/or proline residues. Lengths of these spacers can range from about one to about 5 amino acids. In addition, it is often preferable to physically constrain the targeting molecule by cyclization, which usually results in increased binding. This is usually accomplished by a pair of cysteine residues, flanking the RGD core at a distance of about 4 (having only RGD in between) to 10 amino acids from one another. For example, the pair of cysteine residues, flanking the RGD core can be at a distance of 7 amino acids from one another.
133. Thus, a typical targeting molecule would have the following structure:
-XRGDYX- wherein X is zero to five amino acids and Y is a one or two amino acids, selected from cysteine, serine, threonine and methionine, hi a particularly useful embodiment, X is comprised of glycine residues, but optionally contains at least one, and typically one or two, free thiol- or amine-containing amino acids and/or a single hydrophobic amino acid. Thiol- containing residues include methionine and cysteine; amine-containing residues include lysine and (at least one additional) arginine; and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine. 134. Targeting molecules which bind specifically to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides bear the signal sequence based on Asn-Gly-Arg (NGR). Accordingly, sequences that bind certain integrins can serve as useful targeting molecules to endothelial cells and other cells of the neo-vasculature. For example, the targeting molecule can be an NGR targeting sequence.
135. Non-structural spacers may be a feature of the targeting molecule. Such spacers typically comprise glycine and/or proline residues. Lengths of these spacers can range from about one to about 5 amino acids, hi addition, it is often preferable to physically constrain the targeting molecule by cyclization, which usually results in increased binding. This is usually accomplished by a pair of cysteine residues, flanking the NGR core at a distance of about 4 (having only NGR in between) to 10 amino acids from one another. For example, the pair of cysteine residues, flanking the NGR core can be at a distance of 7 amino acids from one another.
136. Thus, a typical targeting molecule would have the following structure: -XNGRYX- wherein X is zero to five amino acids and Y is a one or two amino acids, selected from cysteine, serine, threonine and methionine. In a particularly useful embodiment, X is comprised of glycine residues, but optionally contains at least one, and typically one or two, free thiol- or amine-containing amino acids and/or a single hydrophobic amino acid. Thiol- containing residues include methionine and cysteine; amine-containing residues include lysine and (at least one additional) arginine; and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine.
137. The targeting molecule can also be a molecule that interacts with a tumor antigen. The tumor antigen can be selected from the list consisting of human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/ρmell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM, alpha- fetoprotein (AFP), COl 7-1 A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm's tumor antigen- 1, telomerase, and melanoma gangliosides. 138. The targeting molecule can also be an aptamer or an antibody specific for the target. Traditionally, the identification of biomarkers and development of antibodies for their specific targeting has been a difficult and time-consuming process that does not always provide the best result for a particular application. For example, although many cancer related biomarkers have been identified, only few of them have shown promising results for cancer screening and prognosis, and it has been recognized that it may be true that only combinations of these biomarkers can provide the best discrimination between cancerous and normal tissue.
139. Numerous reviews have been written about the practice and products of in vitro selection of aptamers. (Conrad, R. C, L. Giver, et al. (1996). "In vitro selection of nucleic acid aptamers that bind proteins." Methods Enzymol 267: 336-67; Osborne, S. E., I. Matsumura, et al. (1997). "Aptamers as therapeutic and diagnostic reagents: problems and prospects." Curr Opin Chem Biol 1(1): 5-9; Famulok, M. and G. Mayer (1999). "Aptamers as tools in molecular biology and immunology." Curr Top Microbiol Immunol 243: 123-36; Hesselberth, J., M. P. Robertson, et al. (2000). "In vitro selection of nucleic acids for diagnostic applications [In Process Citation]." J Biotechnol 74(1): 15-25.). The methods of the present invention may utilize aptamers with unique or improved binding characteristics to a target that is unique to or over represented (as compared to a normal or non-target cell) in, around or on a cell of interest. An "aptamer" as used herein refers to a nucleic acid that binds a target molecule through interactions or conformations other than those of nucleic acid annealing/hybridization described herein. Methods for making and modifying aptamers, and assaying the binding of an aptamer to a target molecule may be assayed or screened for by any mechanism known to those of skill in the art (see for example, U.S. Pat. Nos. 6,111,095, 5,861,501, 5,840,867, 5,792,613, 5,780,610, 5,780,449, 5,756,291 5,631,146 and 5,582,981; as well as PCT Publication Nos. WO92/14843, WO91/19813, and WO92/05285, each of which is incorporated herein by reference).
140. Aptamers are single- or double-stranded DNA or single-stranded RNA molecules that recognize and bind to a desired target molecule by virtue of their shapes. See, e.g., PCT Publication Nos. WO92/14843, WO91/19813, and WO92/05285. The SELEX procedure, described in U.S. Pat. No. 5,270,163 to Gold et al., Tuerk et al. (1990) Science 249:505-510, Szostak et al. (1990) Nature 346:818-822 and Joyce (1989) Gene 82:83-87, can be used to select for RNA or DNA aptamers that are target-specific. In the SELEX procedure, an oligonucleotide is constructed wherein an n-mer, preferably a random sequence tract of nucleotides thereby forming a "randomer pool" of oligonucleotides, is flanked by two polymerase chain reaction (PCR) primers. The construct is then contacted with a target molecule under conditions which favor binding of the oligonucleotides to the target molecule. Those oligonucleotides which bind the target molecule are: (a) separated from those oligonucleotides which do not bind the target molecule using conventional methods such as filtration, centrifugation, chromatography, or the like; (b) dissociated from the target molecule; and (c) amplified using conventional PCR technology to form a ligand- enriched pool of oligonucleotides. Further rounds of binding, separation, dissociation and amplification are performed until an aptamer with the desired binding affinity, specificity or both is achieved. The final aptamer sequence identified can then be prepared chemically or by in vitro transcription.
141. The length of a random sequence tract can range from 20 to over 150 residues, and can be even longer if multiple, random oligonucleotides are combined into a single pool by ligation or other methods. (Bartel, D. P. and J. W. Szostak (1993). "Isolation of new ribozymes from a large pool of random sequences [see comment]." Science 261(5127): 1411-8.). The number of individuals in a random sequence population is typically at least 10.sup.13 and can easily be over 10.sup.15. For most pools, this means that upwards of all possible 25-mers are present, and a proportionately smaller number of motifs longer than 25. Because of the redundancy of biological sequences, the sequence diversity of most random sequence pools likely rivals the sequence diversity of the Earth's biosphere.
142. Aptamers have been selected against a surprising range of targets, ranging from ions to small organics to peptides to proteins to supramolecular structures such as viruses and tissues. (Famulok, M. and G. Mayer (1999). "Aptamers as tools in molecular biology and immunology." Curr Top Microbiol Immunol 243: 123-36; Xu, W. and A. D. Ellington (1996). "Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope." Proc Natl Acad Sci U S A 93(15): 7475-80; Weiss, S., D. Proske, et al. (1997). "RNA aptamers specifically interact with the prion protein PrP." J Virol 71(11): 8790-7; Convery, M. A., S. Rowsell, et al. (1998). "Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution." Nat Struct Biol 5(2): 133-9; Homann, M. and H. U. Goringer (1999). "Combinatorial selection of high affinity RNA ligands to live African trypanosomes." Nucleic Acids Res 27(9): 2006-14.). La particular, aptamers have been selected against a wide variety of proteins, including many nucleic acid binding proteins, such as T4 DNA polymerase (Tuerk, C. and L. Gold (1990). "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science 249(4968): 505-10.) and HIV-I Rev, (Giver, L., D. Bartel, et al. (1993). "Selective optimization of the Rev-binding element of HIV-I." Nucleic Acids Res 21(23): 5509-16.) and multiple non-nucleic acid binding proteins, hi general, anti-protein aptamers seem to recognize basic patches on protein surfaces. For example, the arginine-rich motifs (ARMs) of many viral proteins are recognized by aptamers (reviewed in Ellington, A. D., F. Leclerc, et al. (1996). "An RNA groove [news]." Nat Struct Biol 3(12): 981-4.), the phosphate- binding pockets of both kinases (Conrad, R., L. M. Keranen, et al. (1994). "Isozyme-specific inhibition of protein kinase C by RNA aptamers." J Biol Chem 269(51): 32051-4.) and phosphatases, (Bell, S. D., J. M. Denu, et al. (1998). "RNA molecules that bind to and inhibit the active site of a tyrosine phosphatase." J Biol Chem 273(23): 14309-14.) and the heparin-binding sites on many surface proteins and cytokines, such as basic fibroblast growth factor (Jellinek, D., C. K. Lynott, et al. (1993). "High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding." Proc Natl Acad Sci U S A 90(23): 11227- 31; Jellinek, D., L. S. Green, et al. (1995). "Potent 2'-amino-2'-deoxypyrimidine RNA inhibitors of basic fibroblast growth factor." Biochemistry 34(36): 11363-72.) and vascular endothelial growth factor. (Jellinek, D., L. S. Green, et al. (1994). "Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor." Biochemistry 33(34): 10450-6; Green, L. S., D. Jellinek, et al. (1995). "Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor." Chem Biol 2(10): 683-95.).
143. Aptamers also seem to have an affinity for pockets or cusps on protein surfaces, such as the combining sites of antibodies (Tsai, D. E., D. J. Kenan, et al. (1992). "In vitro selection of an RNA epitope immunologically cross-reactive with a peptide." Proc Natl Acad Sci U S A 89(19): 8864-8) or the active sites of enzymes. (Tuerk, C, S. MacDougal, et al. (1992). "RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase." Proc Natl Acad Sci U S A 89(15): 6988-92.). Almost all proteins have either surface pockets or basic patches (indeed, even proteins with negative pi's, such as T4 DNA polymerase, typically contain sites that can elicit aptamers). Most aptamer: target complexes have dissociation constants in the nanomolar range. Moreover, aptamers recognize their targets with high specificity, and can typically discriminate between protein targets that are highly homologous or differ by only a few amino acids. (Conrad, R., L. M. Keranen, et al. (1994). "Isozyme-specific inhibition of protein kinase C by RNA aptamers." J Biol Chem 269(51): 32051-4; Eaton, B. E., L. Gold, et al. (1995). "Let's get specific: the relationship between specificity and affinity." Chem Biol 2(10): 633- 8; Hirao, L, M. Spingola, et al. (1998). "The limits of specificity: an experimental analysis with RNA aptamers to MS2 coat protein variants." MoI Divers 4(2): 75-89.).
144. As used herein, the term "antibody" encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-I, IgG-2, IgG-3, and IgG-4; IgA-I and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
145. The term "variable" is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.
146. As used herein, the term "antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain integrin binding activity are included within the meaning of the term "antibody or fragment thereof." Specifically, an antibody or a fragment thereof that maintains alphaVbeta3 binding activity is included within the meaning of the term "antibody or fragment thereof. Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
147. Also included within the meaning of "antibody or fragments thereof are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference. 148. Optionally, the antibodies are generated in other species and "humanized" for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, hi general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
149. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species, hi practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. 150. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and Chothia et al., J. MoL Biol., 196:901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
151. It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a disclosed method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994).
152. Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in hnmuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)). The techniques of Cote et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991)).
153. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
154. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988). In a hybridoma method, a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. For example, the immunizing agent can be selected from the list consisting of human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, gpl00/pmell7, GD2, GD3, GM2, TF, sTn, MAGE-I, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV- 16, MUM, alpha-fetoprotein (AFP), CO 17- 1 A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm's tumor antigen-1, telomerase, the vitronectin receptor (αvβ3), CA125 (MUC 16), CD4, CD20, CD30 (Siglec-2), CD30, TNFRSFl), CD33 (Siglec-3), CD52 (CAMPATH-I), CD56 (NCAM), CD66e (CEA), CD80 (B7-1), CD140b (PDGFRβ), CD152 (CTLA4), CD227 (PEM, MUCl, mucin-1), EGFR (HERl, ErbBl), EpCam, GD3 ganglioside, HER2 (HER2/neu, ErbB2), PSMA, Sialyl Lewis, VEGF and melanoma gangliosides. Traditionally, the generation of monoclonal antibodies has depended on the availability of purified protein or peptides for use as the immunogen. More recently DNA based immunizations have shown promise as a way to elicit strong immune responses and generate monoclonal antibodies. In this approach, DNA-based immunization can be used, wherein DNA encoding a portion of integrin expressed as a fusion protein with human IgGl is injected into the host animal according to methods known in the art (e.g., Kilpatrick KE, et al. Gene gun delivered DNA- based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor. Hybridoma. 1998 Dec;17(6):569-76; Kilpatrick KE et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5 microg of DNA. Hybridoma. 2000 Aug;19(4):297-302, which are incorporated herein by referenced in full for the the methods of antibody production) and as described in the examples.
155. An alternate approach to immunizations with either purified protein or DNA is to use antigen expressed in baculovirus. The advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems. Use of this system involves expressing domains of an integrin antibody as fusion proteins. The antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the integrin antibody nucleotide sequence. This results in the display of the foreign proteins on the surface of the virion. This method allows immunization with whole virus, eliminating the need for purification of target antigens. 156. Generally, either peripheral blood lymphocytes ("PBLs") are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, "Monoclonal Antibodies: Principles and Practice" Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. Immortalized cell lines can be those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Other immortalized cell lines can be murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md.
Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal Antibody Production Techniques and Applications" Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against integrin. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RJA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art, and are described further in the Examples below or in Harlow and Lane "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York, (1988).
157. After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
158. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
159. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Optionally, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for integrins and another antigen- combining site having specificity for a different antigen.
160. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab')2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.
161. The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab1 fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
162. An isolated immunogenically specific paratope or fragment of the antibody is also provided. A specific immunogenic epitope of the antibody can be isolated from the whole antibody by chemical or mechanical disruption of the molecule. The purified fragments thus obtained are tested to determine their immunogenicity and specificity by the methods taught herein, hnmunoreactive paratopes of the antibody, optionally, are synthesized directly. An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the antibody amino acid sequence.
163. One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N. Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer- Verlag Inc., NY. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.
164. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (JLrB) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al.,
J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
165. Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
166. Also disclosed are fragments of antibodies which have bioactivity. The polypeptide fragments can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system. For example, one can determine the active domain of an antibody from a specific hybridoma that can cause a biological effect associated with the interaction of the antibody with integrin. For example, amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity. For example, in various embodiments, amino or carboxy-terminal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays. In another example, a fragment of an antibody comprises a modified antibody wherein at least one amino acid has been substituted for the naturally occurring amino acid at a specific position, and a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified antibody. For example, a modified antibody can be fused to a maltose binding protein, through either peptide chemistry or cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide. The hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164.). Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well.
167. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antigen. (Zoller MJ et al. Nucl. Acids Res. 10:6487-500 (1982).
168. A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding. The binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980). Methods of making the compositions
169. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
Nucleic acid synthesis
170. For example, the nucleic acids can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Bcuta et ah, Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et ah, Methods En∑ymoh, 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et ah, Bioconjug. Chem. 5:3-7 (1994). Peptide synthesis
171. A particular advantage of the useful peptides of this invention is that they are readily synthesized by solid phase methods and a variety of combinations are possible to achieve specifically required results. An advantage of the use of solid phase techniques is that the product can be directly synthesized with the C-terminus amidated or otherwise blocked, which is beneficial in forming the procytotoxins of the invention.
172. One method of producing the disclosed proteins, such as (melittin) SEQ ID NO: 6, is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N. Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer- Verlag Inc., NY (which is herein incorporated by reference at least for material related to peptide synthesis). Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.
173. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
174. Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)). 175. Another method of producing the disclosed proteins, such as (melittin) SEQ ID NO: 6, is is to use recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. DNA encoding the the proteins can be readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce the protein, to obtain the synthesis of the protein in the recombinant host cells.
Pharmaceutical Compositions 176. Also provided, in achieving this objective of the invention, are pharmaceutical compositions that, in general, contain a disclosed procytotoxin and a pharmaceutically suitable excipient
177. Another aspect of the present invention is a pharmaceutical composition comprising one or more procytotoxins of the invention and a pharmaceutically suitable carrier or excipient.
178. While compositions of the present invention can be administered, alone, to a patient, it is also possible to administer the compositions in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 179. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is herein incorporated by reference. 180. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular target as described above as well as below.
181. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062- 2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). hi general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). 182. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
Pharmaceutically Acceptable Carriers
183. The compositions described herein can be used therapeutically in combination with a pharmaceutically acceptable carrier.
184. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
185. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
186. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
187. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
188. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
189. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
190. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, pills, powders, granules or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can also be used. 191. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
192. Since the procytotoxins of the invention are amphoteric they may be utilized as free bases, as acid addition salts or as metal salts. The salts must, of course, be pharmaceutically acceptable, and these will include metal salts particularly alkali and alkaline earth metal salts, suitably potassium or sodium salts. A wide variety of pharmaceutically acceptable acid addition salts are available. These include those prepared from both organic and inorganic acids, for example, the pharmaceutically acceptable acid addition salt can be mineral acids. Typical acids which may be mentioned by way of example include citric, succinic, lactic, hydrochloric and hydrobromic acids. Such products are readily prepared by procedures well known to one skilled in the art. 193. Furthermore, the compositions of the present invention can also be modified to have a longer clearance rate and therefore increase bioavailability by protecting the composition from an immune response and other clearance mechanisms afforded by the subject. Indeed, WO 9/2802 discloses that PEGylated compounds exhibit reduced immunogenicity and antigenicity, and circulate in the bloodstream considerably longer than unconjugated proteins. For example, PEG (polyethyleneglycol) polymer chains can be attached to the prolactin variant/prolactin receptor antagonist by methods known in the art, such as by the PEGylation procedure described in Wang et al, Advanced Drug Delivery Reviews; 54 (2002) 547-570, which is incorporated herein by reference.
194. But other agents which can prolong elimination half-life of the therapeutic composition of the present invention are known to the skilled artisan and contemplated herein.
195. In addition, the compositions described herein can also be modified with hydroxyethylstarch (HES). HES is a derivative of naturally occurring amylopektine and is degraded by α-amylase in the body. Methods for making HES-protein conjugates are known in the art. See, for example, EP 1398322, DE 2616086 and DE 2646854, which are incorporated herein by reference.
196. In all such compositions, the cytolytic peptides will normally be the principal physiologically active ingredient. The inventive peptides may be formulated, however, with additional pharmacological agents for combination therapies. When used in treating cancer, for example, they may be formulated with compatible conventional chemotherapeutic agents.
Therapeutic Uses
197. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of a disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
198. Following administration of a disclosed composition, for treating, inhibiting, or preventing tumor formation or tumor growth, the efficacy of the therapeutic composition can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition disclosed herein is efficacious in treating or inhibiting tumor formation or tumor growth in a subject by observing that the composition reduces tumor formation or tumor growth or prevents a further increase in tumor formation or tumor growth. Efficacy of the administration of the disclosed composition may also be determined by measuring the level of tumor specific antigens present in the blood. Methods of measuring the level of tumor specific antigens are well known in the art.
199. The compositions that inhibit tumor formation or tumor growth disclosed herein may be administered prophylactically to patients or subjects who are at risk for tumor formation or tumor growth.
200. Other molecules that interact with tumors to inhibit tumor formation or tumor growth which do not have a specific pharmacuetical function, but which may be used for tracking changes within cellular chromosomes or for the delivery of diagnositc tools for example can be delivered in ways similar to those described for the pharmaceutical products. 201. The disclosed compositions and methods can also be used for example as tools to isolate and test new drag candidates for a variety of cancers.
202. The disclosed compositions and methods can also be used to study cell death. The disclosed composition and methods can also be used to study cell expression of MMP in cells.
203. The disclosed compositions and methods can also be used to study the effect of different cytotoxins on different cell types and also to study the ability of different enzymes to cleave different proteolytic cleavage sites.
Method of Selectively Destroying a Target Cell 204. Also disclosed are methods for selectively destroying a target cell. The disclosed methods typically entail contacting the target cell with a procytotoxin disclosed herein. For example, the procytotoxin can have a cytotoxic peptide bound via a peptide bond to an LAP, wherein the peptide bond is susceptible to cleavage by a target specific protease. Target cells can be cells that are involved in the microvasculature surrounding cancer cells and cancer cells.
205. The procytotoxin of the present invention typically is converted, and thereby activated, into a cytotoxic peptide in a target cell-specific manner by an activity associated with the target cell. In other words, the target cell possesses a specific mechanism for converting the procytotoxin into a cytotoxin. The activated cytotoxic peptide acts on and destroys the target cell in a selective manner. For example, the cell-associated activity can be a protease.
206. Accordingly, a method for selectively destroying a target cell, comprising contacting the target cell with procytotoxin, wherein a proteolytic cleavage site is provided between a latency associated peptide and a cytotoxic peptide, is described. For example, the target cell can be a cancer cell, however any cell that comprises a cell specific protease may be a target.
207. The target cell can have a protease that selectively cleaves the proteolytic cleavage site to which the LAP is attached. Therefore, if the procytotoxin comprises an MMP cleavage site, a target cell with an MMP can cleave the proteolytic cleavage site and "activate" the cytolytic peptide, hi addition, the procytotoxin can comprise a targeting molecule. The targeting sequences can also act as LAPs themselves, as well as direct a procytotoxin to the neo-vasculature and cytoskeletal elements surrounding the target cell. For example, the procytotoxin can comprise a targeting molecule selected from the group consisting of an RGD targeting sequence and an anti-fibronectin ED-B antibody.
208. In one embodiment, the procytotoxin comprises a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is a cytolytic peptide, and wherein the proteolytic cleavage site is an MMP cleavage site, and wherein the LAP comprises the precursor peptide of TGFβ-1, 2, 3, 4, or 5. For example, the procytotoxin can comprise a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is melittin, wherein the proteolytic cleavage site is an MMP2 cleavage site and wherein the LAP is the precursor peptide of TGFβ-1.
209. hi another example, the procytotoxin can comprise a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide, wherein the cytotoxic peptide is an amoebopore, wherein the proteolytic cleavage site is an MMP2 cleavage site and wherein the LAP is the precursor peptide of TGFβ-1. hi either example, a non-target cell, which does not contain the MMP, is unable to activate the protoxin and is thus unaffected by the protoxin.
210. Therapeutic treatment of cancer using the instant cytolytic peptide-based procytotoxin is particularly advantageous because cytolytic peptides are known to be absorbed into the target cell membrane. Even after the target cell is lysed, therefore, the cytolytic peptides are prevented from acting on, and causing undesired destruction of, adjacent non-target cells. For example, see Leippe et al. (1991) Proc. Natl. Acad. Sci. 88: 7659-7663. Moreover, at least in the case where no extra stabilization steps (described above) are taken, it is noted that the instant medicaments are small peptides, which generally will have a fairly short half-life. Hence, even if small amounts of activated cytolytic peptide escapes the surface of the cancer cell, it should be reasonably short-lived and cause little, if any, destruction of non-target cells.
Method for Treating Cancer in a Patient 211. Further disclosed is a method of treating cancer in a patient, hi general, the method comprises administering to a patient a therapeutically effective amount of a procytotoxin. In some embodiments, procytotoxins can be based on an amoebopore, melittin or a cytolytic peptide derived therefrom.
212. Also disclosed is a method for treating a cancer patient, comprising administering a therapeutically effective amount of the pharmaceutical compositions described herein. For example, the dosage of the procytotoxin might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day. For example, the dosage can be from 5 mg/kg to 10 mg/kg. More than one procytotoxin may be administered, and combinations of procytotoxins may also be administered. Optimal delivery routes, dosages and regimens for a given mammalian host can be readily ascertained by one skilled in the art. It will, of course, be appreciated that the actual dose used will vary according to the particular composition formulated, the particular compound used, the mode of application and the particular site, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease.
213. The disclosed compositions can be used to treat any solid tumor. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, or pancreatic cancer.
214. Compounds disclosed herein may also be used for the treatment of precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias. For example, any tissue undergoing angiogenesis which express MMP2 can be targted using the disclosed procytotoxins. 215. A method for treating a cancer patient can comprise administering to a patient a therapeutically effective amount of a procytotoxin comprising a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and a cytotoxic peptide. For example, the cytotoxic peptide can be a pore-forming cytolytic peptide, as described above. In some embodiments, the cytolytic peptide can be a melittin, a melittin analog, or a melittin derivative.
Method of Providing Latency to a Cytotoxin
216. Also disclosed is a method of providing latency to a cytotoxin, comprising covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin.
217. In general the procytotoxin is a cytotoxic peptide that is rendered non-toxic by charge neutralization and/or sterically inhibiting formation of a conformation which renders the peptide toxic. Specifically, the procytotoxin comprises a cytotoxic peptide bound to an LAP via a peptide bond, wherein said peptide bond is susceptible to cleavage by a tumor specific protease. The LAP of the instant invention hinders the cytotoxic peptide from forming an active conformation. For example, when the procytotoxin contacts a target cell, a tumor specific protease cleaves the peptide bond between the LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane.
218. A method of providing latency to a cytotoxin can comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin. The cytotoxin can be a pore-forming cytolytic peptide, as described above. In some embodiments, the cytolytic peptide can be a melittin, a melittin analog, or a melittin derivative. The proteolytic cleavage site can be an MMP cleavage site, and the LAP can comprise the precursor peptide of TGFβ-1, 2, 3, 4, or 5.
219. A method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said cytotoxin is hindered from forming a conformation which renders the peptide toxic, therefore the cytotoxic peptide is rendered non-toxic. 220. A method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide forms a protective shell around the cytotoxic peptide peptide ("caging"), thereby shielding the cytotoxic peptide and hindering, or preventing, its interaction with other molecules in the cell surface or molecules important for its activity. This can be achieved by allowing the LAP to form a dimmer around the cytotoxic peptide, thus forming a protective shell, or cage, around the cytotoxic peptide.
221. A method of providing latency to a cytotoxin can also comprise covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein said latency associated peptide sterically prevents the alpha-helical structure of the cytotoxin from forming. Additionally, the cytotoxic peptide can be modified to include negatively charged amino acids, thereby preventing the toxic pore conformation from forming.
Kits
222. Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include the procytotoxins discussed in certain embodiments of the methods, as well as the pharmaceuticat excipeint and other reagents required to use the procytotoxins as intended. EXAMPLES
223. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
Example 1: Cloning of Melittin Based Procytotoxin Making double-stranded MMP2/melittin template
224. Two complementary oligos, the EcoRI/MMP2/Melittin sense strand (SEQ ID NO: 17) and the EcoRI/MMP2/Melittin anti-sense strand (SEQ ID NO: 18) were synthesized using standard methods. Equal molar ratios of the two oligos were mixed and then heated for five minutes in a one liter beaker with boiled water. The mixture was cooled down on a benchtop to room temperature yielding a double-stranded MMP2/melittin template.
Amplification and subcloning of the double-stranded MMP2/melittin template
225. The double-stranded MMP2/melittin template was amplified by PCR using primer Pl, (SEQ ID NO: 19) and primer P2 (SEQ ID NO: 20) which has the 5' end phosphorylated. PCR was carried out under the following conditions: 94°C for 5 min, then 28 cycles of: 94°C for 30 seconds, 62.50C for 30 seconds and 72°C for 30 seconds; then 72°C for 10 min. The PCR product was then purified using QIAGEN kit (cat. No.: 28104).
226. After purification of the PCR product, the purified product was digested with EcoRI. In addition, a PcR2.1 vector was digested with EcoRI and EcoRV. The EcoRI digested PCR product was then incubated with the EcoRI/EcoRV digested PcR2.1 vector to allow insertion of the digected PCR product into the digested PcR2.1 vector. This resulting plasmid was named pCRMMP2/melittin.
Amplification of the LAP fragment
227. The LAP cDNA fragment was amplified using PCR with plasmid phTGFB-2 as template (ATCC # 59954). A LAP sense primer containing a HindIII site (SEQ ID NO: 21) and a LAP antisense primer containing an EcoRI site (SEQ ID NO: 22) were used in the PCR reaction.
228. PCR was carried out under the following conditions: 94°C for 5 min, then 25 cycles of: 940C for 30 seconds, 75°C for 30 seconds and 72°C for 30 seconds; then 720C for 10 min. The PCR product was then purified using QIAGEN kit (cat. No.: 28104) and digested with HindIII and EcoRI. In parallel, the pCRMMP2/melittin plasmid was also digested with HindIII and EcoRI.
229. The Hindlll/EcoRI digested PCR product was then incubated with the Hindlll/EcoRI digested pCRMMP2/melittin plasmid to allow insertion of the digected PCR product into the digested pCRMMP2/melittin plasmid. This resulting plasmid was named pCRLAP/mmp2/melittin. Mammalian expression vector construction
230. A mouse DHFR cDNA fragment was cut out from plasmid pS V2-DHFR (ATCC# 37146) using BstXl and Not! The ρIRES2-EGFP plasmid was also digested with BstXl and Notl (Clontech). 231. The BstXl/Notl digested DHFR cDNA fragment and the BstXl/Notl digested pIRES2-EGFP plasmid were then purified. The BstXl/Notl digested DHFR cDNA fragment and the BstXl /Notl digested pIRES2-EGFP plasmid were then incubated together under conditions suitable to allow insertion of the digested DHFR cDNA fragment to insert itself into the digested pIRES2-EGFP plasmid. The plasmid was named as pIRES/DHFR. 232. The LAP/mmp2/melittin fragment was cut out of pCRLAP/mmp2/melittin with HindIII and Xhol and inserted into the pIRES/DHFR plasmid cut with Smal. The resulting mammalian expression vector was named as pLAP/mmp2/melittin/IRES/DHFR.
CHO cell expression
233. CHO/DHFR- (ATCC# 9096) cells were transfected with the pLAP /mmp2/melittin/IRES/DHFR plasmid and selected with G418 in medium containing
DMEM+1.5g/L sodium bicarbonate, O.lmM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, 10% FBS, Ix PSN, and 1000 ug/ml G418.
234. Single colonies were picked from the selection and expanded for expression purpose. 235. For expression induction, the above selected cells were switched into serum free medium supplemented with 500 nM of methotrexate and incubated overnight. The medium was collected for future purification.
Example 2: Cloning of Melittin Based Procytotoxin Generation of LAP-MMP2-Melittin Fusion Protein
236. The LAP-MMP2-Melittin construct was cloned into pIRES-DHFR vector.
This plasmid DNA was then transfected into CHO cells as described in Example 1. Forty- eight hours after transfection, cells were selected with G418 in medium containing DMEM+1.5g/L sodium bicarbonate, O.lmM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, 10% FBS, Ix PSN, and 1000 ug/ml G418. Cells were continuously cultured in G418 media containing 10% FBS. Once G418 selection was complete, the transfected CHO cells were adapted to media containing only 1% FBS. 237. Transfected CHO cells were induced to produce the LAP-MMP2-Melittin fusion protein by increasing the concentration of MTX in culture media from 5OnM MTX to 500 nM MTX. At each MTX concentration, cell lysate was obtained and analyzed via SDS PAGE followed by western analysis with LAP antiserum (Figure 1). Western analysis revealed a protein with the predicted molecular weight of the LAP fusion protein. This protein was the most intense at MTX concentrations of 250nm and 500nm. An intense band is also seen at the 5OnM MTX concentration, but there was also a mixture of other bands.
238. hi order to show that the protein observed in the transfected CHO cells was not just an artifact, lysate was obtained from untransfected CHO cells exposed to 500 nM MTX. Figure 2 shows that this protein is not present in CHO cells that were not transfected with the LAP MMP2 Melittin fusion protein. It can therefore be concluded that the LAP fusion protein is present in lysate obtained from fusion protein transfected cells.
239. The next step was to see if the LAP fusion protein was secreted into the culture media. This was examined by collecting media from transfected cells in the presence of 50OnM MTX. Prior to media collection, cells were exposed to each of the following concentration of MTX; 50nm, 10OnM, 25OnM, and 50OnM, for 48 hours. Culture media was then collected from cells exposed to 500 nM MTX, cellular debris was pelleted, and the supernatant was stored at -20 degrees Celsius. In order to concentrate the amount of fusion protein in the culture media, the media was lyophilized. Lyophilized media was then resuspended in water in a volume approximately 1 OX less than the initial volume. LAP
MMP2 Melittin fusion protein was partially purified from the concentrated culture media by hLAP affinity chromatography using a batch procedure. The sample was then analyzed via native PAGE followed by western blotting with hLAP antiserum (Figure 3). This sample was compared to protein obtained from transfected cell lysate. The same bands were seen for both of these samples on the western.
240. LAP MMP2 Melittin was also purified from culture media via a different method. The sample was lyophilized as described above and then a size exclusion column on the Water's HPLC was used for purification. Figure 4 shows the partially purified protein. Example 3: In Vitro Target Cell Killing
241. In vito functionality of the LAP MMP2 Melittin fusion protein can also be assessed. For example, DU- 145 prostate cancer cells which secrete MMP, 2 can be plated in a 48 well plate on day 0. On day 1 , these cells can be treated with the fusion protein for 3 hours, and then cell lysis will be determined via an LDH (lactate dehydrogenase) assay. The reaction can be visualized by an LDH assay.
242. Additionally or alternatively, purified LAP MMP2 Melittin fusion protein can be incubated overnight at 37 degrees Celsius in the presence or absence of active MMP2. The following day, the treated and untreated samples can be incubated with CHO cells. Cells can be treated for 1 hour and then an LDH assay can be done to determine if the cells have been lysed. Treatment of the fusion protein overnight with active MMP2 can cleave the protein at the MMP2 cutting site and release free melittin. When the MMP2 treated fusion protein is incubated with CHO cells, the cells show increased cell lysis as compared to cells incubated with untreated fusion protein.
Example 4: In Vivo Target Cell Killing
243. Functionality of the LAP MMP2 Melittin fusion protein can also be shown in vivo. For example, C57 female mice can be inoculated s.c. with approximately 0.8 million Bl 6 cells. 7 days after tumor inoculation, mice can be injected Lm. with 50ug of LAP MMP2 Melittin plasmid DNA. Mice can then be injected two additional times per week. Tumor size is reduced and longevity of the mice is increased.
244. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Claims

CLAIMSWhat is claimed is:
1. A procytotoxin comprising a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and the cytotoxic peptide.
2. The procytotoxin of claim 1 , wherein the cytotoxic peptide is a cytolytic peptide.
3. The procytotoxin of claim 2, wherein the cytolytic peptide is selected from the group
consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter vcoccus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin oϊAeromonas
hydrophila, esculentin, granulysin, haemolysin of Vibrio parahaemolyticus,
intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1,
neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4,
NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming
cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
4. The procytotoxin of claim 3, wherein the cytolytic peptide is an amoebapore.
5. The procytotoxin of claim 4 comprising SEQ ID NO: 10 or SEQ ID NO: 12.
6. The procytotoxin of claim 3, wherein the cytolytic peptide is a melittin.
7. The procytotoxin of claim 6 comprising SEQ ID NO: 2 or SEQ ID NO: 4
8. The procytotoxin of claim 1 , wherein the latency associated peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4, or 5.
9. The procytotoxin of claim 1 , wherein the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site.
10. The proteolytic cleavage site of claim 9, wherein the proteolytic cleavage site is a MMP2 cleavage site.
11. A pharmaceutical composition, comprising the procytotoxin of claim 1, and a
pharmaceutically acceptable excipient.
12. A method of treating cancer in a patient comprising administering to said patient a therapeutically effective amount of a procytotoxin comprising a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided
between the latency associated peptide and cytotoxic peptide.
13. The method of claim 12, wherein the cytotoxic peptide is a cytolytic peptide.
14. The method of claim 13, wherein the cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore,
amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E,
barbatolysin, cytolysin of Enterococcus faecalis, delta hemolysin, diphtheria toxin,
El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas
hydrophila, esculentin, granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin of Streptococcus intermedins, the lenti virus lytic peptide, leukotoxin
of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated
lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4,
NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming
cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
15. The method of claim 14, wherein the cytolytic peptide is an amoebapore.
16. The method of claim 14, wherein the procytotoxin comprises SEQ ID NO: 10 or SEQ ID NO: 12
17. The method of claim 14, wherein the cytolytic peptide is a melittin.
18. The method of claim 14, wherein the procytotoxin comprises comprising SEQ ID NO: 2 or SEQ ID NO: 4.
19. The method of claim 12, wherein the latency associated peptide comprises the
precursor peptide of TGFβ-1, 2, 3, 4, or 5.
20. The method of claim 12, wherein the proteolytic cleavage site is a matrix
metalloproteinase (MMP) cleavage site.
21. The method of claim 20, wherein the proteolytic cleavage site is a MMP2 cleavage site.
22. The method of claim 12, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's
Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer,
head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin
cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat,
larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, or pancreatic cancer.
23. A method of providing latency to a cytotoxin comprising covalently linking a latency associated peptide and a proteolytic cleavage site with a cytotoxin, wherein
said latency associated peptide and proteolytic cleavage site provides latency to said cytotoxin.
24. The method of claim 23, wherein the cytotoxic peptide is a cytolytic peptide.
25. The method of claim 24, wherein the cytolytic peptide is selected from the group
consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E,
barbatolysin, cytolysin of Enterococcus faecalis, delta hemolysin, diphtheria toxin,
El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticns, intermedilysin of Streptococcus intermedius, the lentivirus lytic peptide, leukotoxin
of ' Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated
lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium
perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming
cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
26. The method of claim 25, wherein the cytolytic peptide is an amoebapore.
27. The method of claim 25, wherein the procytotoxin comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 12
28. The method of claim 25, wherein the cytolytic peptide is a melittin.
29. The method of claim 25, wherein the procytotoxin comprises the sequence of comprising SEQ ID NO: 2 or SEQ ID NO: 4.
30. The method of claim 23, wherein the latency associated peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4, or 5.
31. The method of claim 23, wherein the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site.
32. A method of selectively destroying a target cell that is a cancer cell, comprising contacting the target cell with a procytotoxin, which comprises a latency associated
peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and a cytotoxic peptide, wherein said latency associated peptide acts to prevent the procytotoxin from forming a lytically active conformation.
33. The method of claim 32, wherein the cytotoxic peptide is a cytolytic peptide.
34. The method of claim 33, wherein the cytolytic peptide is selected from the group
consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore,
amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enterococcus faecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas
hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin
of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4,
NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
35. The method of claim 34, wherein the cytolytic peptide is an amoebapore.
36. The method of claim 34, comprising SEQ ID NO: 10 or SEQ ID NO: 12.
37. The method of claim 34, wherein the cytolytic peptide is a melittin.
38. The method of claim 34 comprising SEQ ID NO: 2 or SEQ ID NO: 4.
39. The method of claim 32, wherein the latency associated peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4, or 5.
40. The method of claim 32, wherein the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site.
41. The method of claim 40, wherein the proteolytic cleavage site is a MMP2 cleavage site.
42. A method of selectively destroying a target cell that is involved in the microvasculature surrounding cancer cells, comprising contacting the target cell with
a procytotoxin, which comprises a latency associated peptide and a cytotoxic
peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and a cytotoxic peptide, wherein said latency associated peptide acts to prevent the procytotoxin from forming a lytically active conformation.
43. The method of claim 42, wherein the cytotoxic peptide is a cytolytic peptide.
44. The method of claim 43, wherein the cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E, barbatolysin, cytolysin of Enter vcoccus faecalis, delta hemolysin, diphtheria toxin,
El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticus, intermedilysin of Streptococcus intermedins, the lenti virus lytic peptide, leukotoxin
of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment I5 neokyotoφhin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming
cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
45. The method of claim 44, wherein the cytolytic peptide is an amoebapore.
46. The method of claim 44, comprising SEQ ID NO: 10 or SEQ ID NO: 12.
47. The method of claim 44, wherein the cytolytic peptide is a melittin.
48. The method of claim 44 comprising SEQ ID NO: 2 or SEQ ID NO: 4.
49. The method of claim 42, wherein the latency associated peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4, or 5.
50. The method of claim 42, wherein the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site.
51. The method of claim 50, wherein the proteolytic cleavage site is a MMP2 cleavage site.
52. A method of selectively destroying a target cell that is a MMP2 producing cancer
cell, comprising contacting the target cell with a procytotoxin, which comprises a latency associated peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency associated peptide and a cytotoxic peptide, wherein said latency associated peptide acts to prevent the procytotoxin from
forming a lyrically active conformation.
53. The method of claim 52, wherein the MMP2 producing cancer cell is a MMP2
producing tumor microvascular endothelial cell.
54. The method of claim 52, wherein the cytotoxic peptide is a cytolytic peptide.
55. The method of claim 54, wherein the cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-lE, brevinin-2E,
barbatolysin, cytolysin of Enterococcus faecalis, delta hemolysin, diphtheria toxin,
El Tor cytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysin of Vibrio par ahaemolyticus, intermedilysin of Streptococcus intermedins, the lentivirus lytic peptide, leukotoxin
of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1,
neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin, and streptolysin, analogs of the pore-forming cytolytic peptide, and derivatives of the pore-forming cytolytic peptide.
56. The method of claim 55, wherein the cytolytic peptide is an amoebapore.
57. The method of claim 55, comprising comprising SEQ ID NO: 10 or SEQ ID NO: 12.
58. The method of claim 55, wherein the cytolytic peptide is a melittin.
59. The method of claim 55 comprising SEQ ID NO: 2 or SEQ ID NO: 4.
60. The method of claim 52, wherein the latency associated peptide comprises the
precursor peptide of TGFβ-1, 2, 3, 4, or 5.
61. The method of claim 52, wherein the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site.
2. The method of claim 61, wherein the proteolytic cleavage site is a MMP2 cleavage
site.
EP06825387A 2005-10-04 2006-09-29 Latent procytotoxins and uses thereof Withdrawn EP1943350A4 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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* Cited by examiner, † Cited by third party
Title
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