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WO1997033908A1 - Peptides lytiques - Google Patents

Peptides lytiques Download PDF

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Publication number
WO1997033908A1
WO1997033908A1 PCT/AU1997/000160 AU9700160W WO9733908A1 WO 1997033908 A1 WO1997033908 A1 WO 1997033908A1 AU 9700160 W AU9700160 W AU 9700160W WO 9733908 A1 WO9733908 A1 WO 9733908A1
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WIPO (PCT)
Prior art keywords
peptide
terminal
amphipathic
peptide according
helix
Prior art date
Application number
PCT/AU1997/000160
Other languages
English (en)
Inventor
Donald Edward Rivett
Peter John Hudson
Jerome Anthony Werkmeister
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to NZ331771A priority Critical patent/NZ331771A/xx
Priority to AU19170/97A priority patent/AU723904B2/en
Priority to EP97906936A priority patent/EP0901502A4/fr
Priority to JP53212397A priority patent/JP2001517201A/ja
Publication of WO1997033908A1 publication Critical patent/WO1997033908A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to unique peptides having lytic activity, the lytic ability of which may be
  • peptides are designed to be able to form amphipathic helixes. They may either be based on known sequences of natural products which form these structures, or may be designed from first principles using theoretical predictions of the sequences required to form such
  • the lytic peptides of the invention may be targeted to specific cells, for example by linking to a targeting moiety such as an antibody.
  • amino acid and “residue” are used herein as synonyms.
  • cytotoxic amphipathic peptides include insect venoms such as melittin from bee venom, the magainins from frog skin, cecropins, bombolitins, mastoparans and related peptides, and the ⁇ -haemolysin from Staphylococcus aureus .
  • the cytopathic amphipathic peptides are generally basic, but vary widely in size and function. Melittin is 26 amino acids long, and is strongly cytotoxic and haemolytic;
  • magainins are 23 amino acids long, are poorly haemolytic, and are toxic to bacteria.
  • the bombolitins and mastoparans are only 14 amino acids long; they are cytotoxic, but have only weak haemolytic activity.
  • cytotoxic amphipathic peptides have been extensively studied, particularly melittin and the magainins, and many of the structural requirements for cytolytic function are now understood.
  • a melittin analogue with an ideal amphipathic ⁇ -helical sequence from positions 1-19, followed by the natural sequence from positions 20-26, has been synthesised; this polypeptide was more active than naturally-occurring melittin, showing that the haemolytic activity of this protein does not require the kink in the ⁇ -helix (DeGrado et al, J. Am. Chem. Soc, 1981 103 679-681; Dempsey et al, FEBS Letters, 1991 281 240-244).
  • Melittin constructs with the reverse sequence (16-26) (1-13) retain their
  • WO96/18104 by Torvey Pines Institute for Molecular Studies (Blondell et al) discloses methods for synthesis and screening of large numbers of melittin analogues having anti-microbial, haemolytic or catalytic achorly. The entire disclosure of WO96/18104 is
  • Lys 7 can be replaced by serine without
  • cytotoxic amphipathic peptides particularly melittin
  • melittin have been a target for intensive study because of their potential use as immunotoxins, when linked to
  • Therapeutic uses are expected to include cancer treatment, treatment of infections and other
  • the peptides of the invention may be used to form immunotoxins or the like, or as antibiotics.
  • the peptides may also be used as biosensors.
  • peptides comprising an amphipathic ⁇ -helix having no lytic activity may be activated by adding a group which neutralises the negative charge of the carboxy terminal residue. They have also found that by adding a positively charged amino acid to the amino
  • removal of negative charge may act to cause an overall change in the net charge of the peptide and thereby render it lytically active.
  • removal of negative charge may just act locally at the carboxy terminus rendering the peptide lytically active.
  • the inventors have also surprisingly found that the presence of an amino acid sequence or other group at the amino terminal of an otherwise lytically active peptide comprising an amphipathic ⁇ -helix, renders the peptide lytically inactive when the amino acid sequence or other group decreases the positive charge of the peptide.
  • inactive peptide can be activated by dimerisation.
  • the invention provides a peptide with lytic activity, having an amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically, wherein the N-terminal and/or C- terminal of said peptide comprises one or more moieties which result in an increased positive charge compared to the charge of a peptide of identical amino acid sequence and structure but not comprising said moiety.
  • the peptide may be naturally occurring or non- naturally occurring.
  • non-naturally occurring peptide refers to a peptide in its entirety which does not occur in nature. Notwithstanding this the peptide may comprise fragments or parts which are based on natural sequences.
  • amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically means a peptide with a comparable function to that of the amphipathic ⁇ -helix of native melittin. This will generally be in the form of an ⁇ -helix which is about 20 amino acids in length.
  • the peptides are particularly preferred.
  • including the ⁇ -helix are between 21 and 26 residues in length.
  • the ⁇ -helix may be derived from naturally- occurring cytopathic amphipathic peptides, including melittin and other insect venoms, such as wasp, ant, or scorpion venom, antibacterial peptides such as a magainin, a cecropin, a bombolitin, or a mastoparan, or another known cytopathic amphipathic peptide.
  • dimers they may be composed of ⁇ -helices which are the same or
  • amphipathic peptides may comprise a naturally-occurring sequence as described above, or portion thereof, together with an artificial sequence, as described herein.
  • amphipathic ⁇ -helical sequences with positively charged residues periodically located along the peptide chain are known, and in some cases are several-fold more active than melittin. Fragments having this type of sequence may be used in the invention, either alone or as extensions of naturally occurring sequences.
  • amino acids which make up the peptide of the invention may be naturally occurring amino acids, or non-naturally occurring analogues or homologues thereof, for example phenylglycine, norleucine or homoarginine. Such amino acids will be well known to those skilled in the art, for example as listed in WO96/18104. In one particularly preferred embodiment where the peptide is 21 amino acids or less the ⁇ -helix has at least 45% amino acid homology to native melittin.
  • Preferred amino acids are Gly 1, Gly 3, Ala 4, Thr 10, Gly 12, Ser 18 & Trp 19 or Phe 19.
  • amino acids at positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 16, 17, 19 and 20 are Hydrophobic amino acids at positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 16, 17, 19 and 20.
  • amino acid at position 14 is Pro or hydroxyproline.
  • positive charge may be amino acids or other chemical groups which are positively charged themselves, or which block negative charges which would otherwise occur on the peptide if the moiety was not present.
  • the increase in the positive charge may be an increase in the overall positive charge of the peptide, or a localised increase in positive charge. The latter may also have the effect of activating or increasing lytic activity.
  • the peptide of the invention is in the form of an isolated preparation which has been purified, to at least some degree.
  • the invention provides a non-naturally occurring peptide with lytic activity, having an amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically, wherein the C-terminal residue of said peptide is not negatively charged and wherein absence of a negative charge at the C-terminal residue is provided by blocking the negative charge of said residue.
  • not negatively charged means that the amino acid has a positive charge or is neutral.
  • Blocking the negative charge of the C-terminal residue may be achieved by any convenient means such as by addition of a neutral or positive substituent.
  • Suitable substituents include -NH 2 ,
  • acetamidomethyl ACM
  • the C- terminal residue is a cysteine residue which has been neutralised.
  • the cysteine residue may be substituted with ACM.
  • the invention provides a lytic peptide, comprising two amphipathic ⁇ -helices which are not themselves lytic, linked to form a dimer which has lytic activity.
  • the dimer may be a homodimer or a heterodimer.
  • the nature of the link between the two components of the dimer is not critical; one convenient linkage is via a disulphide bond, which may be effected by having a cysteine or cysteine-amide residue at the C-terminal of each of the components of the dimer, or via a bridge moiety such as -CH 2 S(CH 2 ) n SCH 2 -, where n is 1 to 4.
  • the link moiety be able to hold the two peptide chains together.
  • the link is of approximately the same size as a disulphide bond, but the person skilled in the art will recognise that many other linkages are possible.
  • the disulphide link is particularly convenient, as it can be directly expressed, using recombinant
  • CH 2 groups there may be a bridging unit of CH 2 groups between the end of the peptide chain and the thiol group which is to be oxidised.
  • a length of CH 2 chain which will be functionally active may readily be identified by routine testing
  • ⁇ -helical sequences with positively charged residues periodically located along the peptide chain are known, and in some cases are several-fold more active than melittin. Fragments having this type of sequence may be used in the invention, either alone or as extensions of naturally occurring sequences.
  • an alternative embodiment of the first aspect of the invention provides a cytotoxic dimer of an amphipathic peptide, in which the amphipathic peptide has the general formula:
  • ⁇ glycine; a hydrophobic amino acid including but not limited to alanine, isoleucine, leucine, valine, and norleucine; or ⁇ ;
  • ⁇ asparagine, glutamine, serine or threonine
  • ⁇ phenylanine, tryptophan, tyrosine, a non-naturally occurring aromatic amino acid, or a synthetic aromatic residue
  • X represents hydrophilic amino acids, at least one of which is a basic amino acid such as lysine, arginine or homoarginine; preferably two or more of the hydrophilic amino acids are basic amino acids.
  • R is arginine
  • Z is an amino acid which is not negatively charged, or is either a cross-linking agent, such as a
  • heterobifunctional linking group or a blocking group
  • Z when Z is a blocking group it may be -NH 2 , ACM or the like.
  • C or Z may be at either end of the peptide, but not at both ends.
  • an aromatic residue designated ⁇ is beneficial for function .
  • this is at residue 19 , as in Peptide 01B .
  • Either a synthetic aromatic residue or a non-naturally occurring aromatic amino acid may be used as alternatives to naturally occurring aromatic amino acids . See for example U . S . Patent No . 5294605 by Scripps
  • the cross-link which forms the dimer may be at either the C-terminal or the N-terminal end of the peptide. While terminal cysteine or lysine residues are particularly convenient for formation of the cross-link, as discussed above the skilled person will be aware of alternative means of linking the two peptide chains to form a dimer. For example, a cross-link at the C-terminal end, as in
  • peptide 01B can be achieved using 1-ethyl-3-(3- dimethylaminopropyDcarbodiimide hydrochloride (EDC) in conjunction with a suitable diamine, such as
  • the length of the bridge between the two peptide chains is relatively flexible, and the disulphide can be replaced by a -CH 2 S (CH 2 ) n S CH 2 -crosslink (where n is 1-4), by a lysine, by small lengths of peptide, or by other suitable chemical moieties.
  • These peptides may be amidated, and/or acetylated at the N-terminal.
  • a typical example is based on melittin, which has the following sequence:
  • a typical peptide is synthesised containing the first 20 residues of the melittin sequence, with the addition of a cysteine-amide residue as residue 21 in the sequence.
  • This peptide is oxidised to form the dimer as a disulphide.
  • Potentially any sequence which forms an amphipathic helix could be used, provided that certain key residues are present.
  • the helical dimer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl dimer
  • the peptides of the invention may be synthesised, using conventional methods such as solid phase synthesis, particularly if a C-terminal amide such as a cysteine amide is desired.
  • the dimeric peptide can be directly synthesised by forming peptide bonds at both the ⁇ -NH 2 and the ⁇ -NH 2 groups of lysine, ie. lysine can form a branch point.
  • they may be expressed using recombinant DNA technology; this is of course not suitable for C-terminal amides, but this modification could be introduced subsequently.
  • the means of production may be selected depending on the use for which the peptide is intended.
  • the peptide of the invention may be attached to a carrier molecule, either by chemical linking or by
  • the monomeric form of a peptide of the invention may be expressed in a host cell, for example as a fusion protein with a fusion protein carrier, and then either the peptide- carrier hybrid may be dimerised, or if the peptide is to be in the form of a heterodimer, a suitable second half of the dimer may be added either by chemical linkage or by
  • fusion protein carrier is an Fab fragment of an antibody
  • one peptide of the invention could be expressed fused to the heavy chain and the second peptide of the invention
  • the peptide may be linked to a cleavable affinity or detection label; several suitable labels are known in the art, for example FLAGTM (U.S. Patent No. 4,703,004), T7-Tag and HSV-Tag (Novagen), and BTag (our International Patent Application
  • the carrier molecule is selected from the group consisting of an antibody, an Fab, Fv, ScFv or other antigen-specific fragment or analogue thereof, epidermal growth factor (EGF) and transforming growth factor- ⁇ (TGF- ⁇ ).
  • the antibody, analogue or fragment is or is derived from a monoclonal antibody it may be modified to reduce immunogenicity, eg. by "humanization". Suitable methods are well known in the art.
  • the antibody is specific for a cell surface antigen and more preferably is specific for a cancer-specific antigen. In one embodiment this antigen is specific for a solid tumour. Many such antigens are known in the art, such as
  • gastrointestinal and lung carcinomas prostate carcinoma antigen, ovarian carcinoma antigen, and breast carcinoma antigen.
  • the invention provides a peptide which is capable of lytic activity, comprising an amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically, and
  • the cleavable moiety may be any moiety which can be linked to the ⁇ -helix and inactivates lytic function.
  • the moiety is an amino acid sequence such as a pre-sequence Acetyl- AVY-, Acetyl-SSGYSNT- or Acetyl-AVE-.
  • the cleavable sequence is selectively removable by enzymatic means.
  • Suitable groups for blocking the negative charge may be any group without a negative charge which does not otherwise adversely affect the lytic ability of the peptide.
  • Suitable groups include other amphipathic ⁇ - helical peptides, particularly those able to form a dimer with the peptide capable of lytic activity.
  • Other suitable groups include -NH 2 and ACM. While -NH 2 does not preclude dimer formation where the terminal residue is cysteine, ACM, which binds the thiol group prevents dimer formation.
  • the present invention provides a method of modulating the lytic activity of a peptide, said peptide comprising an amphipathic ⁇ -helix of
  • step (a) results in an increase of lytic activity or activation of lytic activity and step (b) results in a decrease or inactivation of lytic activity.
  • modulating means changing or altering the lytic ability of the peptide and includes changing a lytically inactive peptide into a lytically active peptide and the reverse.
  • the moieties which increase or decrease the positive charge may be those described earlier.
  • binding to the N and/or C terminal refers to chemically linking the moiety to one of the available groups on the terminal residue.
  • removing from the N and/or C terminal refers to cleaving or otherwise chemically removing the moiety in question from the remainder of the peptide.
  • under suitable conditions means carrying out the method in the presence of the appropriate chemicals, buffers, temperature, for an appropriate time.
  • the invention provides a method of activating a lytically inactive peptide, wherein said inactive peptide comprises an amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically and
  • said method comprising, -under suitable conditions, treating said peptide with
  • the cleaving agent may be any suitable agent to remove the cleavable moiety.
  • the cleavable moiety is an amino acid sequence the cleaving agent may be an
  • the blocking agent may be any agent suitable for blocking the negative charge.
  • the blocking agent may be another amphipathic ⁇ -helical peptide which contains a cysteine residue and is capable of forming a dimer.
  • Other suitable blocking agents may be the suitable groups described above.
  • under suitable conditions in relation to the cleaving agent and/or blocking agent means treating the peptide under appropriate reaction conditions and for an appropriate time such that the cleaving agent and/or blocking agent can perform their desired functions.
  • suitable conditions will be determined by the nature of the peptides and the agents used and will be well known to those skilled in the art.
  • the invention provides a method of inactivating a lytically active peptide wherein said peptide comprises an amphipathic ⁇ -helix of sufficient length and character to allow the peptide to function lytically, and/or a non-negatively charged carboxy terminal residue, and where said peptide is optionally in the form of a dimer comprising two ⁇ -helices, said method
  • the cleavable moiety is an amino acid presequence which reduces the overall positive charge of the peptide.
  • the invention provides a pharmaceutical composition, comprising a peptide of the invention, either alone or coupled to a carrier molecule, together with a pharmaceutically-acceptable carrier.
  • the composition may also comprise an antibiotic or a toxic anion, such as fluoride ion, or an anti-tumour, antibacterial, anti-viral or antiparasite agent.
  • the invention provides a method of treatment or alleviation of cancer or of a manifestation of infection with human immunodeficiency virus , comprising the step of administering an effective amount of a peptide of the invention, either alone or coupled to a carrier molecule, to a subject in need of such treatment.
  • the invention provides a method of treatment of a microbial infection, comprising the step of administering an effective amount of a peptide of the invention to a subject in need of such treatment, said dimer comprising an amphipathic helical sequence of the invention and a cecropin or a cecropin-like peptide.
  • the invention provides a biosensor, comprising a first monomeric form of a peptide of the invention linked to an antigen and a second monomeric form of a peptide of the invention linked or adsorbed to a solid support or to a membrane, a
  • Dimerisation may be detected by membrane lysis or by ion-channel formation.
  • the dimeric form of the molecule constitutes an voltage-gated ion channel, so that dimerisation results in current flow.
  • dimerisation is effected via formation of a disulphide bond, so that the biosensor of this aspect of the invention provides a redox switch.
  • Figures 1 to 6 show flow cytometric analysis of the effect of cytolytic peptides on CEM T-cell lymphoma cells. The cells were treated with the peptides for
  • Figure 1A shows flow cytometric analysis of untreated control cells, with side scatter (vertical axis) plotted against forward scatter (horizontal axis),
  • Figure 1B shows the viability of control cells, as measured using exclusion of propidium iodide; cell count (vertical axis) is plotted against propidium iodide
  • Figure 2 shows the effect of intact melittin on cells
  • Figure 3 shows the effect of peptide 29
  • Figure 4 shows the effects of peptide 01B
  • Figure 5 shows the effects of peptide 10
  • Figure 6 shows the effects of peptide 34
  • Figure A shows flow cytometric analysis
  • Figure B shows cell viability
  • Figure 7 shows the effects of peptide AP11
  • Figure 8 shows the effect of melittin on cell membrane potential, as measured by formation of ion
  • Figure 8A shows control cells
  • Figure 8B shows melittin-treated cells.
  • FIG. 9 shows diagrammatic representations of immunotoxin molecules constructed according to the
  • Figure 9a depicts an immunotoxin in which the inactivating leader peptide is joined by a proteolytic cleavage sequence (X) to a cytolytic peptide joined to a scFv shown here as VH-VL.
  • X proteolytic cleavage sequence
  • Figure 9b depicts an immunotoxin in which the chemically or genetically conjugated Fab comprising one heavy chain fused to a proteolytically sensitive linker sequence (X) fused to a cytolytic peptide and a second light chain molecule fused to another proteolytically sensitive linker sequence (X) fused to another cytolytic peptide.
  • the genetic conjugation is in the description of figure 10.
  • Figures 9c and 9d depict reverse orientation forms of 9a and 9b.
  • Figure 10 shows the specific oligonucleotides used to construct an scFv expression cassette for the Ncol- EcoRI restriction sites of pGC. The PCR cloning strategy is also shown using these oligonucleotides. The genetic conjugation of scFv or Fab to cytolytic peptide and
  • proteolytic cleavage signal and construction of expression cassettes use PCR and cloning techniques that are published (Coia G., Hudson, P.J. and Lilley G.G. (1996) Journal of Immunological Methods. 192, 13-23).
  • Norleucine was inserted into peptides 10 and 34.
  • the lysine branched dimer, peptide 30, was synthesised by the technique described above, but
  • FmocLys (Fmoc)OH was used at the C-terminal. This yielded a crude product which consisted of a mixture of deletion peptides along with the desired peptide which was purified by C 18 reverse phase HPLC.
  • the monomeric cysteine peptides 01b and 42C were prepared by alkylating peptides 01B and 42 with iodoacetic acid in dimethylformamide, after prior reduction with tributylphosphine.
  • acetylated peptides 01C, 42B, 44B and 46B were obtained by treating the parent peptides with an excess of acetic anhydride in dimethylformamide containing a trace of triethylamine.
  • phenylalanine can be substituted for tryptophan without loss of activity.
  • Example 1 was compared with that of melittin. Peptides were dissolved in dimethyl sulphoxide (DMSO) at 5 mg/ml and serially titrated by two-fold
  • phosphate buffered saline PBS
  • concentrations in the 96-well U-bottomed microtitrs plates ranged from 200 ⁇ g/ml to 0.7 ⁇ g/ml. 0.6% suspension of washed human red blood cells (lOO ⁇ l) were added for 1 hour. Plates were centrifuged at 150 X g for 5 min, and 100 ⁇ l aliquots were transferred to a 96-well polyvinyl chloride plate (Dynatech Laboratories,
  • Haemolysis was assessed by measurement of optical density at 405nm with an automatic EAR 400 SF ELISA plate reader (SLT Lab instruments, Groedig/Salzburg,
  • Table 1 summarises the haemolyic effects of all peptides after 1 hour at various concentrations ranging from 200 ⁇ g/ml to 0.7 ⁇ g/ml.
  • Peptide 10 is based on the sequence published by Degrado et al ((1981)
  • peptide 10 has a similar lytic activity to melittin (Table 1). Likewise peptide 34, which has no sequence identity with melittin has only a slightly reduced activity, somewhere around a 4-fold reduction.
  • Peptide 01B the truncated (C-terminus-deficient) analogue of melittin was found to be highly active causing significant haemolysis at concentrations as low as 12 ⁇ g/ml.
  • Peptide 41 the truncated (C-terminus-deficient) analogue of the lytic peptide 34, was completely inactive even at the highest concentration.
  • Substitution of lysine and proline into this sequence at positions 7 and 14 respectively resulted in significant lytic activity, albeit still around 4-fold less than peptide 34 on a molar basis. Both substitutions were necessary since replacement with only lysine at position 7 (peptide 44) resulted in only a mild lytic activity.
  • Replacement at position 7 with arginine in association with substitution of proline at position 14) produced a highly lytic peptide (peptide 46, Table 1) which was equally if not more active than peptide 42.
  • the haemolytic activity of the resulting peptides required a free unacetylated N-terminus (peptides 42C, 01C, 42B, 44B and 46B.
  • scattering of laser light from cells moving in a fluid stream to give information about the state of the cells.
  • Light scattered in the direction of the laser beam is measure of cell size.
  • Light scattered at right angles to the beam is an indication of granularity within the cell, and hence of cell membrane integrity.
  • fluorescent molecules can be added to the cells, and their specific fluorescence when excited by the laser beam can be measured by light emission at right angles to the laser beam. This fluorescence can give information about the composition or state of the cells, depending on the specific fluorescent dye used.
  • concentration of peptide (lOO ⁇ g/ml) was chosen from the haemolytic titration curves.
  • Flow cytometry was carried out on a Coulter EPICS® Elite flow cytometer with illumination at 488nm. Forward light scatter was measured at 488nm and propidium iodide fluorescence was detected at greater than 600nm.
  • membrane potential and indicate the formation of ion channels after addition of peptides (lOO ⁇ g/ml) for 5 minutes.
  • cells in PBS, were pre-loaded with 0.05 ⁇ M DiSc 3 in normal saline for 15 min. at room temperature. Flow cytometry was carried out as above with illumination at 488nm for light scatter and at 633nm for excitation of DiSC 3 fluorescence.
  • Fluorescence of the DiSC 3 was measured at greater than 633nm. Because the fluorescence emission spectra of propidium iodide and DiSC 3 overlap (Schapiro (1994) op cit), the two dyes could not be used simultaneously.
  • Figure 1 results with intact melittin are shown in Figure 2.
  • Figures 3, 4, 5, 6 and 7 respectively show results with peptide 29, peptide 01B, peptide 10,
  • the dots on the diagram in A for each figure are measurements on individual cells, and their density
  • Figure 1A shows a normal cell population. It can be seen that the measurements of most cells lie in an area to the bottom centre of the diagram. Dead or damaged cells have measurements in the bottom to middle left of the diagram. A shift from the normal cell area toward the left or top of the figure is an indication of cell damage.
  • Figure IB shows normal cells that are not
  • Carbocyanine dyes are fluorescent probes which dissolve in cell membranes and give a fluorescent signal which is dependent on the orientation of the dye molecules within the cell membranes. This orientation is dependent on the voltage difference across the cell membrane, and therefore the intensity of the fluorescent signal is proportional to the voltage drop.
  • Cells that have been affected by cytolytic peptides develop ion channels that decrease the cell membrane potential, resulting in a shift of the fluorescence intensity histogram towards the left of the diagram.
  • DiSC 3 carbocyanine dye DiSC 3
  • Peptides capable of lytic activity in accordance with the second aspect of the invention may be produced in accordance with the following. While the following relates to dimers it will be appreciated that monomeric peptides may be similarly produced.
  • inactivating presequence may be added during peptide synthesis or the relevant nucleic acids encoding it may be included in the construct if the peptide is being made by recombinant means. It should be noted that phenylalanine may be substituted for tryptophan with only minimal loss of lytic activity.
  • the length of the bridge between the two peptide chains is relatively flexible and the disulphide can be replaced by either a - CH 2 S (CH 2 ) x S CH 2 - (where x is 1-4) crosslink or a lysine or small lengths of peptide or other chemical moiety.
  • the presequences are added as required and their sequence being dependent on the enzyme which is planned to cleave at the appropriate site.
  • Acetyl-A V Y- Prostate Specific Antigen or Chymotrypsin
  • Acetyl- S S G Y S N T - PSA or Chymotrypsin
  • Acetyl-A V E- Staphylococcus aureus V8 protease, Houmard, J. & Drapsau, G. R. (1972), Proc.Natl.Acad.Sci.USA,
  • proline 14 where its inclusion enhances activity of the dimer, but removal of proline from melittin has either no effect or even a positive enhancement of activity, peptides 10 and 34 (Degrado et al (1983) Biophys. J. 37: 329-338; Werkmeister et al (1993) Biochim. Biophys. Acta 1157:50-54.
  • Figure 9 depicts cartoon diagrams of
  • Figure 10 describes specific oligonucleotides which were synthesised and used to construct an scFv (as shown in Figure 9a) as an expression cassette in the Ncol- EcoRl site of vector pGC. (Coia et al, J. Immunol Methods, 1996 192 13-23).
  • the scFv was derived from hybridoma 1C3 (glycophorin specific; Coia et al, 1996).
  • the recombinant immunotoxin was purified
  • PSA prostate-specific antigen
  • Figure 9a depicts an immunotoxin in which an inactivating leader peptide is joined by a proteolytic cleavage sequence (X) to a cytolytic peptide joined to an scFv (in this case in the orientation VH-VL).
  • X proteolytic cleavage sequence
  • Figure 9b depicts a chemically or genetically conjugated Fab comprising one heavy chain fused to a cytolytic peptide and a second light chain molecule fused to another proteolytically sensitive linker sequence fused to another cytolytic peptide.
  • the details of methods of genetic construction will be known to persons skilled in the art from previous descriptions such as Dolezal et al, (Immunotechnology, 1995, 1 197-219). It should be
  • Figures 9c and d depict reverse orientation forms of Figures 9a and 9b.
  • peptides can be used in a wide range of applications which exploit the property that the peptides have little or no biological activity either in the
  • one lytically inactive monomeric form of the peptide is attached to an antigen and a second is attached or adsorbed to a solid support or to a membrane, a receptor, a bound antibody or a Fab fragment, the measurement being as a result of membrane lysis or by ion-channel formation.
  • the dimeric form of the molecule is attached to an antigen and a second is attached or adsorbed to a solid support or to a membrane, a receptor, a bound antibody or a Fab fragment
  • the peptide is attached, by means of a suitable cross-linking agent, either as a monomer or as a dimer, to an antibody or Fab fragment, Fv fragment or ScFv fragment or other antigen-specific
  • the antibody is directed to a target antigen which does not mediate internalization, more preferably a carcinoma-specific antigen.
  • a target antigen which does not mediate internalization, more preferably a carcinoma-specific antigen.
  • the peptide of the invention is expressed either as a monomer or as a
  • immunotoxin can be utilised as the monomer, or can be dimerised prior to utilisation.
  • the monomeric peptide is crosslinked to the immunoglobulin or Fab fragment, with the same or different monomer attached thereto.
  • One monomer chain is attached to the protein and the second free peptide crosslinked to it, preferably via the formation of a disulphide.
  • a third alternative is to couple a peptide of the invention to a growth factor, such as epidermal growth factor or transforming growth factor- ⁇ , either chemically or as a fusion protein, which would enable the peptide of the invention to target cells bearing receptors for these growth factors.
  • a growth factor such as epidermal growth factor or transforming growth factor- ⁇
  • the peptides of the invention can overcome the problem of a cytolytic molecule killing the cell in which it is produced, by expressing half of the dimer, and then for example oxidising this at the target site to form the active molecule.
  • Other methods will readily occur to the person skilled in the art.
  • a peptide of the invention can be expressed with one or more tyrosine residues incorporated directly adjacent to or near to the peptide sequence, which are then cleaved by the target cell. While such cleavage may not be essential for activity, activity could be increased in this way.
  • prostate-specific antigen has protease activity which induces specific cleavage at tyr-tyr (see PCT/AU95/00536 entitled "Assay for the
  • amphipathic helical sequence Using two different designed sequences for each helical segment it is possible to produce a dimer which will lyse bacterial cells but not mammalian cells.
  • amphipathic helical sequence Using two different designed sequences for each helical segment it is possible to produce a dimer which will lyse bacterial cells but not mammalian cells.
  • amphipathic helical sequence For example the amphipathic helical sequence:
  • cecropins may be coupled to a suitable segment from a class of peptides known as cecropins.
  • cecropins Some cytolytically- and antibacterially-active cecropin A - melittin hybrid peptides are known (Diazachirica et al, Eur. J. Biochem., 1994 224 257-263; Fernandez et al, Biopolymers, 1994 34 1251-1258).
  • a disulphide such a segment may suitably be:
  • cysteine residue may be replaced by a penicillamine residue in one of these peptides.
  • peptide sequences are only examples, and potentially any amphipathic helical sequence could be substituted for the first peptide and likewise several alternative sequences derived from the cecropins could be substituted for the second peptide.
  • peptides have little or no biological activity until activated by an appropriate enzyme.
  • the peptide In cancer treatment the peptide is designed to be released from its inactive form in the presence of a tumour specific protease, such as PSA.
  • a tumour specific protease such as PSA.
  • an appropriate enzyme can be delivered to the site of a tumour by an antibody prior to treatment with the peptide.
  • the pre-toxin is attached, by means of a suitable cross-linking agent, either as a monomer or as a dimer to an antibody or Fab fragment.
  • a suitable cross-linking agent either as a monomer or as a dimer to an antibody or Fab fragment.
  • the monomeric peptide is expressed with the Fab fragment.

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Abstract

L'invention concerne un peptide à activité lytique, ayant une hélice α amphiphatique d'une longueur suffisante et d'une nature appropriée, pour permettre au peptide d'avoir une activité lytique. L'extrémité N-terminale et/ou l'extrémité C-terminale de ce peptide comprend un ou plusieurs groupes qui produisent une augmentation de la charge positive du peptide par rapport à la charge du peptide ayant la même séquence d'aminoacides et la même structure, mais ne comprenant pas ce ou ces groupes. L'invention concerne, également, des procédés d'activation et d'inactivation de la fonction lytique, des compositions pharmaceutiques et des méthodes de traitement.
PCT/AU1997/000160 1996-03-13 1997-03-13 Peptides lytiques WO1997033908A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
NZ331771A NZ331771A (en) 1996-03-13 1997-03-13 Lytic peptides with amphipathic alpha helices and positively charged N- or C-terminal, methods of activation/inactivation, compositions and methods of treatment are also included
AU19170/97A AU723904B2 (en) 1997-03-13 1997-03-13 Lytic peptides
EP97906936A EP0901502A4 (fr) 1996-03-13 1997-03-13 Peptides lytiques
JP53212397A JP2001517201A (ja) 1997-03-13 1997-03-13 溶解性ペプチド

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPN8614 1996-03-13
AUPN8614A AUPN861496A0 (en) 1996-03-13 1996-03-13 Lytic peptides

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WO1997033908A1 true WO1997033908A1 (fr) 1997-09-18

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041535A3 (fr) * 1997-03-18 1998-12-23 Anmat Technology Limited Activation de peptides
WO2000002909A3 (fr) * 1998-07-09 2000-05-04 Univ Southern California Peptides amphiphiles isoles derives de la queue cytoplasmique de proteines de l'enveloppe virale
WO2000029433A3 (fr) * 1998-11-18 2000-10-05 Tolin As Produit
US6723830B2 (en) 1998-09-25 2004-04-20 Children's Medical Center Corporation Short peptides which selectively modulate the activity of protein kinases
AU781677B2 (en) * 1999-11-12 2005-06-02 Syntaxin Limited Use of lytic toxins and toxin conjugates
US7094750B2 (en) 2000-05-09 2006-08-22 Greenville Hospital System Therapeutic pore-forming peptides
US7595173B2 (en) 2002-04-22 2009-09-29 Dow Global Technologies Inc. Low-cost production of peptides
US7713927B2 (en) * 2007-01-16 2010-05-11 The Regents Of The University Of California Antimicrobial peptides
US8936916B2 (en) 2008-02-14 2015-01-20 3M Innovative Properties Company Polypeptides for microbial detection
CN105121472A (zh) * 2012-10-30 2015-12-02 埃斯佩兰斯医药公司 抗体/药物缀合物及其使用方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018104A1 (fr) * 1994-08-24 1996-06-13 Torrey Pines Institute For Molecular Studies Polypeptides apparentes a la melittine, leurs ensembles de melanges et banques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018104A1 (fr) * 1994-08-24 1996-06-13 Torrey Pines Institute For Molecular Studies Polypeptides apparentes a la melittine, leurs ensembles de melanges et banques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, (1991), Vol. 30, S.E. BLONDELLE and R.A. HOUGHTON, "Hemolytic and Anti-Microbial Activities of Twenty-Four Individual Omission Analogues of Melittin", pages 4671-4678. *
BIOPHYSICAL JOURNAL, Volume 37, No. 1, (January 1982), W.F. DE GRADO et al., "Kinetics and Mechanism of Hemolysis Induced by Melittin and by a Synthetic Melittin Analogue", pages 329-338. *
See also references of EP0901502A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041535A3 (fr) * 1997-03-18 1998-12-23 Anmat Technology Limited Activation de peptides
WO2000002909A3 (fr) * 1998-07-09 2000-05-04 Univ Southern California Peptides amphiphiles isoles derives de la queue cytoplasmique de proteines de l'enveloppe virale
US6723830B2 (en) 1998-09-25 2004-04-20 Children's Medical Center Corporation Short peptides which selectively modulate the activity of protein kinases
US6770626B2 (en) 1998-09-25 2004-08-03 Children's Medical Center Corporation Tissue remodeling
WO2000029433A3 (fr) * 1998-11-18 2000-10-05 Tolin As Produit
US7422740B1 (en) 1999-11-12 2008-09-09 Health Protection Agency Use of lytic toxins and toxin conjugates
AU781677C (en) * 1999-11-12 2006-09-07 Syntaxin Limited Use of lytic toxins and toxin conjugates
AU781677B2 (en) * 1999-11-12 2005-06-02 Syntaxin Limited Use of lytic toxins and toxin conjugates
US7094750B2 (en) 2000-05-09 2006-08-22 Greenville Hospital System Therapeutic pore-forming peptides
US7425535B2 (en) 2000-05-09 2008-09-16 Ghc Research Development Corporation Therapeutic pore-forming peptides
US7595173B2 (en) 2002-04-22 2009-09-29 Dow Global Technologies Inc. Low-cost production of peptides
US7713927B2 (en) * 2007-01-16 2010-05-11 The Regents Of The University Of California Antimicrobial peptides
US8609608B2 (en) 2007-01-16 2013-12-17 C3 Jian, Inc. Antimicrobial peptides
US8936916B2 (en) 2008-02-14 2015-01-20 3M Innovative Properties Company Polypeptides for microbial detection
CN105121472A (zh) * 2012-10-30 2015-12-02 埃斯佩兰斯医药公司 抗体/药物缀合物及其使用方法
US10233214B2 (en) 2012-10-30 2019-03-19 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

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AUPN861496A0 (en) 1996-04-04
ZA972186B (en) 1997-11-10
CA2248782A1 (fr) 1997-09-18
EP0901502A4 (fr) 2001-09-26
EP0901502A1 (fr) 1999-03-17

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