CA2248782A1 - Lytic peptides - Google Patents

Abstract

The invention provides a peptide with lytic activity, having an amphipathic .alpha.-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. Methods of activation to provide activity and for inactivation of lytic activity, pharmaceutical compositions, and methods of treatment are described.

Description

W097/33~ PCT/AU97/001 ~YTIC rK~ S

This invention relates to unique peptides ha~ing lytic activity, the lytic ability of which may be activated or inactivated.
The~e peptides are de~igned to be able to form amphipathic helixes. They may either be based on known se~nce~ of natural products which form the~e structure~, or may be designed from first ~rinciple~ u~ing theoretical prediction~ of the ~e~nceR required to form such ~tructure~. The lytic peptide~ of the invention may be targeted to ~pecific cell~, for example by l;nkin~ to a targeting moiety ~uch ac an antibody.

R~c~ ,~Ql~ND OF THE lNVL~ ON
In general, throughout this specification the stAn~~rd three letter and single letter codes for amino ac~ds are used. The terms "amino acid" and "residue" are used herein as synonyms.
Many li~ing organism~ are able to produce and secrete toxic compounds. Of these, snake, spider and insect venoms are perhaps the best known. One important class of naturally-produced toxic compounds is the cytotoxic amphi~athic peptide~. These include insect venoms such as melittin from bee venom, the m~g-;n;n~ from frog skin, cecropin~, bombolitins, mastoparans and related peptide~, and the ~-haemolysin from Staphylococcus aureu~.
The cytopathic amphipathic peptides are generally ba~ic, but vary widely in ~ize and function. Melittin is 26 amino acids long, and is strongly cytotoxic and haemolytic;
ma~;n;n~ are 23 amino acids long, are poorly haemolytic, and are toxic to bacteria. The bombolitina and mastoparans are only 14 amino acids long; they are cytotoxic, but have - only weak haemolytic acti~ity.
The cytotoxic amphipathic peptide~ have been exten~ively ~tudied, particularly melittin and the RECEIVED 2 JA.

magainins, and many of the structural requirements for cytolytic function are now understood. For exam~le, a melittin analogue with an ideal am~hipathic a-helical ~equence from ~ositions 1-19, followed by the natural sequence from ~ositions 20-26, has been synthesised; this ~oly~e~tide was more active than naturally-occurring melittin, showing that the haemolytic activity of this ~rotein does not require the kink in the a-helix (DeGrado et al, J. Am. Chem. Soc., 1981 103 679-681; Dem~sey et al, FEBS Letters, 1991 281 240-244). Melittin constructs with the reverse sequence (16-26)(1-13) retain their antibacterial activity, but show no haemolytic activity (Boman et al, FEBS Letters, 1989 259 103-106). Many othe~
analogues of melittin have been synthesised, and it is suggested that some residues are essential, such as Lys~
and Tr~lg, and that the ~rinci~al requirement for lytic activity is a large a-helical am~hi~athicity (Blondelle and Houghten, Biochemistry, 1991 30 4671-4678; Pe~tide Res., 1991 4 12-18; Blondelle et al, Biochim. Biophys. Acta., 1993 1202 331-336); a hydro~hobic: hydro~hilic ratio of at least 2:1 and a strongly positively-charged C-terminal region a~ears also to be essential (DeGrado et al, Biophys. J. 1982 37 329-338).
WO96/18104 by Torrey Pines Institute for Molecular Studies (Blondelle et al) discloses methods for synthesis and screening of large numbers of melittin analogues having anti-microbial, haemolytic or catalytic activity. The entire disclosure of WO96/18104 i8 incor~orated herein by thi~ cross-reference. This document discloses monomeric analogues of melittin, in which lys is ~referably re~laced by glu or a~p, more ~re$erably glu.
More recently we have found that'arginine re~idues are essential for the biological activity of melittin, while ly~ine i~ minimally important. In ~articular, ~ys, can be re~laced by ~erine without significantly affecting activity, and thu~ a~pear~ only to \\lSlSLLOl\hom~S\Lui~ p\~p ci~ iro-pct-~u97-00160 doc 21/01/98 ~MENDED SHEET
IPEA/AU

be important in ma~n~A~n;ng the amphipathic helix (Werkmei~ter et al, Biochem. Biophy~. Acta., 1993 1157 50-64).
In addition to studie~ on characteristic~ of naturally-occurrin~ lytic peptide~, ~ynthetic, highly amphipathic a-helice~ con~i~ting only of ~eu and ~y~
re~idues in a 2:1 ratio have been syntheRi~ed. Ranging from 12-22 re~idue~ long, these peptiaes were mainly a-helical, and were hi~hly haemolytic, with activity varying according to the ~eptide length. The 15-, 20-, and 22-residue long peptide~ were 5 to 10 timeR ~ re active than melittin, and more active than magainin or mastoparan by ~everal order~ of magnitude (Cornut et al, F~BS Letter~, 1994 349 29-33).
The cytotoxic am~hipathic peptideR, particularly melittin, have been a target for ;nten~ive ~tudy becau~e of their potential u~e a~ immunotoxin~, when linked to antibodie~ a~ targeting agents (Dunn et al, Im~unotechnology 1996 2 229-240). However, hitherto ~uch application~ have been limited, hecA~e the pe~tide~ are toxic to normal cells a~ well as to the target cell~. In addition, in the ca~e of melittin there i8 the risk of ~evere allergic ~ide-effects. It appearR that the characteri~tic clu~ter of ~y~ and Arg re~idue~ at the C-terminal, which i~ required for the activity of melittin (Schroder et al, Ex~erientia, 1971 27 764-765); ~Ah~rmann~
in "Natural Toxins" D. Eaker and T. Wa~trom, Pergamon Presa, New York 1980 173-181) i~ also re~pon~ible for the~e allergic side-effect~.
In work leA~;ng up to the present invention the inventor~ have found mean~ of activating potentially lytic but inactive peptide~. They have also found means of inactivating lytic peptide~. The~e fin~ing~ are import_nt becau~e they have implications for therapeutic u~es of peptides who~e lytic abilitie~ can be activated and inactivated. Therapeutic uQes are expected to include cancer treatment, treatment of infections and other condition~ where it iR deRirod to kill cell~. The ~eptide~
of the invention may be u~ed to form immunotoxins or the like, or as antibiotic~. The peptides may al~o be u~ed as bio~en~or~.
The inventor~ have ~urpri~ingly di~covered that peptides compri~ing an amphipathic a-helix having no lytic activity (or reduced lytic activity) may be activated by A~;ng a group which neutrali~e~ the negative charge of the carboxy terminal reRidue. They have al~o found that by ~;ng a positively charged amino acid to the amino terminal of an amphipathic a-helix thiR contribute~ to increaQing lytic activity. Without w;~hi n~ to be bound by theory it appears that the negative charge on the terminal re~idue in a number of ~eptide~ ~ro~ce~ by the inventors i~ re~onsible for the~e proteins l~ck;ng lytic activity or having r~ ce~ lytic activity. It appears that neutrali~ation of the negative char~e activates the inactive ~eptide. Thu~ removal of negative charge may act to cause an overall change in the net charge of the peptide and thereby render it lytically active. Alternatively, removal of negative charge may ju~t act locally at the carboxy terminus rendering the peptide lytically active.
The inventor~ have also ~urpri~ingly found that the pre~ence of an amino acid ~equence or other group at the amino terminal of an otherwi~e lytically active peptide comprising an amphi~athic ~-helix, renderQ the peptide lytically inactive when the amino acid ~e~uence or other group decrea~es the ~o~itive charge of the ~eptide.
Removal of thi~ amino acid sequence or group renders the peptide lytically active. Alternatively, and even more surpriRingly, the inventors ha~e found that such an inactive peptide can be activated by dimerisation.
It will be a~reciated that variou~ aRpects of the in~ention are a~plicable to conjugate delivery ~ystems, including immunotoxins, to antibacterial agentR, and to biosensor sy~tems.

S ~ LARY OF THE lNVlSN-~lON
In a first aspect the invention ~rovides a peptide with lytic activity, having an amphipathic a-helix of sufficient length and character to allow the peptide to function lytically, wherein the N-terminal and/or C-terminal of said peptide compri es one or more moieties which result in an increased positive charge compared to the charge of a ~eptide of identical amino acid Qequence and structure but not comprising said moiety.
The peptide may be naturally occurring or non-naturally occurring.
The term "non-naturally occurring peptide" refer~
to a peptide in its entirety which does not occur in nature. Notwithst~nA;ng this the pe~tide may comprise fragmentR or part~ which are based on natural sequences.
The term "amphipathic a-helix of sufficient length and character to allow the peptide to function lytically" means a pe~tide with a comparable function to that of the amphi~athic a-helix of native melittin. This will generally be in the form of an a-helix which is about 20 amino acids in length. Preferably the peptides including the a-helix are between 21 and 26 residues in length.
The a-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 a~ a magainin, a cecropin, a bombolitin, or a mastoparan, or another known cytopathic amphipathic peptide. In the case of dimers they may be composed of a-helices which are the same or different. One or both of the amphipathic pe~tides may comprise a naturally-occurring sequence as described above, or portion thereof, together with an artificial sequence, as described herein.

W097/33~8 PCT/AU97/001 Ideally amphipathic a-helical seq~ with ~o~itively charged residues periodically located along the ~eptide chain are known, and in ~ome cases are ~everal-fold more active than melittin. Fragments having this type of ~equence may be u~ed in the in~ention, either alone or as exten~ions of naturally occurring Reql~nces.
The amino acids which make up the peptide of the invention may be naturally occurring amino acid~, or non-naturally occurring analogues or homologues thereof, for example phenylglycine, norleucine or homoarginine. Such amino acidQ will be well known to tho~e ~killed in the art, for example a~ listed in W096/18104. In one particularly preferred embodiment where the peptide i~ 21 amino acid~ or less the a-helix has at lea~t 45% amino acid homology to native melittin.
The inventors' initial re~ults indicated that the following amino acids may be e~sential to the lytic function of the a-helix : Lys or Arg 7 and Pro 14.
Preferred amino acidR are Gly 1, Gly 3, Ala 4, Thr 10, Gly 12, Ser 18 & Trp 19 or Phe 19.
We have subRequently found that the rule~
originally propoQed were far too rigid, and that there i8 in fact very considerable latitude for ~ubstitution of amino acids. We now believe that the following are preferable, if not e~sential:
Hydrophilic amino acids at positions 3, 7, 10, 11, 14 and 18, of which at least two, preferably including the amino acid at position 7, should be ~ys and/or Arg; and Hydrophobic amino acid~ at positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 16, 17, 19 and 20. Ad~antageously the amino acid at ~osition 14 is Pro or hydroxyproline.
The moieties which result in an increased 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 ~resent. The increase in the positive 9 7 ~ n ,~
i~L~~i~J ~

charge may be an increa~e in the overall ~ositive charge of the ~e~tide, or a locali~ed increase in ~ositive charge.
The latter may al~o have the effect of activating or increasing lytic activity.
Preferably the pe~tide of the invention is in the form of an i~olated ~re~aration which ha~ been ~urified, to at lea~t some degree.
In an alternative aa~ect related to the fir~t as~ect, the invention ~rovide~ a non-naturally occurring ~e~tide with lytic activity, having an am~hi~athic a-helix of sufficient length and character to allow the ~e~tide to function lytically, wherein the C-terminal residue of said ~e~tide is not negatively charged and wherein ab~ence of a negative charge at the C-terminal residue is ~rovided by blocking the negative charge of said residue.
The term "not negativeIy charged" means that the amino acid has a ~ositive charge or i8 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 ~ositive substituent. Any convenient group which does not have a negative charge may be em~loyed. Suitable substituents include -NH~, acetamidomethyl (ACM) and the like. Optionally the C-terminal residue i~ a cysteine residue which has its thiol grou~ blocked. For exam~le the cy~teine residue may be substituted with ACM.
In a ~referred embodiment of the first as~ect, the invention provides a lytic ~e~tide, com~rising two am~hi~athic a-helices which are not themselves lytic, linked to form a dimer which has lytic activity.
The dimer may be a ho~ dimer or a'heterodimer.
The nature of the link between the two com~onents of the dimer is not critical, one convenient linkage is via a aisul~hide bond, which may be effected by having a cysteine or cysteine-amide residue at the C-terminal of each of the com~onents of the dimer, or via a bridge ~ iety such as \\~Ol\h~ S\Lui~ C-ep\~ci-\c~iro-pct-~u97-00160 toc 21/01/99 ~MENDED SHEET
IPEA/AU

W097133~8 PCT/AU971~1~

-CH2S(CH2)nSCH2-, where n is l to 4. The only requirement is that the link moiety be able to hold the two peptide C~; nR together. Preferably the link i8 of ap~roximately the same size a~ a disnlrh;~e bond, but the per~on skilled in the art will reco~n;~e that many other linkages are possible. For example, maleic anhyaride or maleic anhydride-like moieties ~uch a~ those de~cribed in Australian Patent Application No. 90098/9l, lysine, short peptiae~, or ly~ine-(am;noc~rroic acid)2-lysine links may be used. The ~killed person will readily be able to identify linkage groups which may be suitable, and to test whether in fact they are able to produce the de~ired effect.
The dis1~l~hi~le link i8 particularly convenient, a~ it can be directly ex~ressed, u~ing recombinant technology, or synthesised, and then simply oxidi~ed under appropriate condition~, which are very well known in the art, to form the link. Optionally, there may be a bridqing unit of CH2 group~ between the end of the ~eptide chain and the thiol group ~rh;ch is to be ox~ ed. A length of CH2 chain which will be functionally acti~e may readily be identified by routine testing As described earlier, ideally amphipathic a-helical se~nce~ with positively charged residues periodically located along the peptide chain are known, and in ~ome ca~e~ are ~everal-fold more active th~n melittin.
Fragment~ having this type of sequence may be uRed in the invention, either alone or as extensions of naturally occurring sequence~.
In addition to the wholly or partly naturally-deri~ed ~e~)~nce~ referred to above, an alternative embodiment of the first aQpect of the invention ~rovides a cytotoxic dimer of an amphipathic peptide, in which the amphipathic ~eptide has the general formula:

n Q X X Q Q X n n X X Q Q (X, P or hypro) n Q n X Q n (C or Z) or (C or Z) Q QXXQQXQ Q X X n Q P n Q n x Q n;

more preferably Qn~Qn(ROrR)QQ~QnPQQQ~n(Corz) or n QQQQQ(E~ or R) n n ~ ~ n n P n n Q ~:~P n (c or Z) or (C or Z) n n ~ ~ QQ (K or R) QQ~;~QQPQQQ~Q
or QQQQQQ (R or R) nn ~- ~ Qn p nnQ ~ ~Q

and whorein the symbols have the following --nings Q = glyclne; a hydrophobic amino acid includlng but not 11m~ted to al~n1ne, i~oleucine, leucine, valine, and norleucine; or ~;
= a~paragine, glutamine, serine or threonine;
= phenylanine, tryptophan, tyrosine, a non-naturally occurring aromatic amino acid, or a synthetic aromatic residue; and X repr-~onts hydrophilic am$no aclds, at leaot one of which i8 a basic amino acid such as lysine, arginine or homoarginine; preferably two or more of the hydrophilic amino acids are basic amino aclds.
R = i~ arglnlne K = lysine hypro = hyd o~y~ oline P = proline C = cysteine Z = is an amino acid which is not negatively charged, or is either a cross-l1nk~ ng agent, such as a c_ ~ cially available heterobifunctional l~nklng group, or a hlock~ng group; when Z i~ a blocking group it may be -NH2 ~ ACM or the like.

C or Z may be at either end of the peptide, but not at both ends.

W O 97/33gO8 PCT/AU97tO0160 Preferred ~equ~ncQs ~re:
C I a A V I~ K V ~ T T a ~ P ~ L I S W I C -~2 (Pept~dQ OlB) C G I G A V K L V ~ T T G L P A ~ I S W I -N~2 (Peptide 29) 2(G I G A V ~ K V L T ~ G ~ P A ~ I S W)R -N~2 (Peptide 30) X L Q S L V S L V Q S L V S ~ V L W F L R S R ~ N N -N~2 (Peptide 34) Q S L V K L V ~ S L V P ~ V L Q F ~ C -N82 (Peptide 42) ~ L Q S ~ V ~ ~ V Q S ~ V S ~ V ~ Q ~ ~ C -N~2 (P~ptide 44) G ~ G A L V ~ ~ V T S G V P ~ V L S W L C -N~2 (Peptide 56) A V R G I G A V L K V L T T G ~ P A L I S W I C -N~2 (Peptid~ 50) lO Ac A V R G I G A V L K V ~ T T G ~ P A L I S W I C -N~2 (Peptide 50A) G I G A V L R V L T T G L P A L I S W I M-NH2 (PeptidQ 64M) S S ~ G I K A V ~ K V L T T G L P A ~ I S W I A-N~2 (Peptlde APll) S S L G I G A V L K V L T T G ~ P A ~ I S W I A-NB2 (Peptlde APl3) where -NH2 indicates the C-t~ 'n~l amide group.

It a~pear~ that an aromatic residue designated is beneficial for function. Preferably this i~ at residue 19, aR in Pe~tide 01B. Either a ~ynthetic aromatic residue or a non-naturally occurring aromatic amino acid may be used as alternatives to naturally occurring aromatic amino acids. See for exam~le ~.S. Patent No. 5294605 by Scri~ps Re~earch Institute.
The cross-link which forms the dimer may be at either the C-terminal or the N-terminal end of the ~e~tide.
While terminal cysteine or lysine residues are particularly convenient for formation of the cross-link, as discussed above the skilled ~er~on will be aware of alternative means of l; n~; ng the two ~e~tide ChA; n~ to form a dimer. For exam~le, a cross-link at the C-terminal end, as in peptide OlB, can be achieved using 1-ethyl-3-(3-dimethylaminopro~yl) CA ~ho~; imide hydrochloride (EDC) in conjunction with a suitable diamine, ~uch as tetramethyl~ne~;Amine (~utre~cine). An alternati~e cross-link at the N-terminal can be effected using a reagent such as 2,3-dibromopropionyl-N-hydroxysuccinimide ester (M~Ren7ie et al, J. Protein Chem., 1988 7 581-592).
The length of the bridge between the two pe~tide ch~;n~ is relatively flexible, and the aisul~hide can be re~laced by a -CH2 S (CH2)nS CH2-crosslink (where n is 1-4), CA 02248782 l998-09-ll W097/33~8 PCTtAU97/00160 by a lysine, by smali len~ths of ~eptide, or by other suitable chemical moieties.
The~e peptides may be amidated, and/or acetylated at the N-terminal.
A typical example is based on melittin, which ha~
the following sequence:

G I G A V L R V ~ T T G ~ P A ~ I S W I R R ~ R Q Q -N~2 The potent lytic acti~ity of melittin is well established (Habermann and Jentsch 1967), as is the loss of activity on removal of residues 21-26 (Schroder et al 1971).
A ty~ical pe~tide is syntheRised containing the first 20 residues of the melittin sequence, with the addition of a cysteine-amide residue as residue 21 in the sequence. ThiR ~eptide is oxidised to form the dimer as a disulphide. Potentially any sequence which fonms an am~hipathic helix could be used, ~rovided that certain key residue are ~re~ent.
In a particularly preferred ~mhodiment of the invention, the helical dimer is .

G I G A V ~ R V ~ T T G ~ P A ~ I S W I C
I

G I G A V ~ R V ~ T T G ~ P A ~ I S W I C

The ~eptides of the in~ention may be _ynthesised, using conventional methodR such as solid phase synthesis, particularly if a C-terminal amide ~uch as a cysteine amide i~ desired. Where the cross-link is ~ia lyRine, the dimeric peptide can be directly synthe~i~ed by forming peptide bond~ at both the a-NH2 and the ~-NH2 qroups of lysine, ie. ly~ine can form a branch point. Alternatively, they may be expressed using recombinant DNA technology;

W097/33~8 PCT/AU97/~1 thiR i~ of cour~e not suitable for C-terminal amide~, but thi~ modification could be introduced Rubsequently. The means of ~roduction may be selected depenA~ng on the use for which the peptide iR intended.
The peptide of the invention may be attAch~ to a carrier molecule, either by chemical linking or by Rynthesi~ aR a fuRion ~rotein using recombinant methods.
The monomeric form of a peptide of the invention may be ex~reRsed in a hoQt cell, for example as a fusion protein with a fu~ion protein carrier, and then either the peptide-carrier hybrid may be dimeri~ed, or if the peptide i~ to be in the form of a heterodimer, a ~uitable ~econd half of the dimer may be added either by chemical linkage or by expres~ion a~ a fu~ion protein to complete the dimeric peptide of the invention. For example, if the fuRion protein carrier iR an Fab fragment of an antibody, one peptide of the invention coula be expre~ed fused to the heavy chain and the ~econd peptide of the invention expressed fused to the light chain of the Fab.
In either ~ynthetic method, the peptide may be linked to a cleavable affinity or detection label; several ~uitable lAh~lB are known in the art, for example FLAGTM
(~.S. Patent No. 4,703,004), T7-Tag and HSV-Tag (Novagen), and BTag (our International Patent Application No. PCT/A~96/00516 entitled "Epitope Tagging System").
Preferably the carrier molecule i~ Relected from the group con~i~ting of an antibody, an Fab, Fv, ScFv or other antigen-~pec~fic fragment or analogue thereof, epidermal ~rowth factor (EGF) and tran~forming growth factor-a (TGF-a). Where the antibody, analogue or fragment i~ or i~ derived from a monoclonal antibody it may be modified to reduce immunogenicity, eg. by "humanization~.
Suitable method~ are well known in the art. Preferably the antibody i~ specific for a cell surface anti~en and more preferably iR Rpecific for a cancer-E~pecific antigen. In one embodiment thi~ antigen i~ ~pecific for a ~olid tumour.

Many such antigens are known in the art, such a~
carcino~mhryonic antigen, which i~ a~sociated with qastrointestinal and lung carcinomas, ~rostate carcinoma antigen, ovarian carcinoma antigen, and breast carcinoma antigen.
In an alternative embodiment related to the first aspect the invention pro~ides a peptide which i~ capable of lytic acti~ity, com~rising an amphipathic a-helix of sufficient length and character to allow the peptide to function lytically, and (a) a cleavable moiety located amino terminal to said a-helix, wherein said moiety ~revents or retards lytic function of said ~e~tide and wherein ~e,..oval of ~aid moiety reRults in a lytically active peptide, and/or (b) a negati~ely-charged carboxy terminal residue of said peptide, wherein the negative charge ~revents or retards lytic function of _aid ~eptide and wherein removal of said charge by blocking with a suitable group results in lytic acti~ity.
The clea~able moiety may be any moiety wh;ch can be linked to the a-helix and inacti~ates lytic function.
Preferably the moiety i~ an amino acid sequence such as a pre-sequence Acetyl- AVY-, Acetyl-SSGYSNT- or Acetyl-AVE-.
Preferably the cleavable Qe~uence i8 selectively removable by enzymatic means.
Suitable groups for blocki ng the negative charge may be any group without a negative charge which does not otherwise adversely affect the lytic ability of the ~eptide. Suitable groups include other am~hipathic a-helical peptides, particularly those able to form a dimer with the peptide capable of lytic activity. Other suitable group~ include -NH2 and ACM. While -NH2 does not preclude dimer formation where the terminal residue i~ cy_teine, ACM, which binds the thiol group prevent~ dimer formation.
In a third aspect ~he pre_ent invention pro~ides CA 02248782 l998-09-ll a method of modulating the lytic activity of a ~e~tide, saia peptide comprising an amphi~athic a-helix of sufficient len~th and character to allow the ~eptide to function lytically, ana optionally com~rising additional amino acids, said method com~rising, under ~uitable conditions, (a) increasing the positive charge of saia peptide by A~; nq to the N and/or C terminal one or more moieties which increa~e the positive charge of the peptide, or (b~ decrea~ing the ~ositive charge of said peptide by removing from the N ana/or C terminal one or more moieties which decrea~e the positive charge of the peptide wherein step (a) results in an increa~e of lytic activity or activation of lytic activity ana step (b) results in a decrease or inactivation of lytic activity.
The term "moaulating" 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 aecrease the positive charge may be those described earlier.
The term "~Aing to the N and/or C terminal~
refers to chemically l;n~;ng the moiety to one of the available group~ on the terminal resiaue. The term ~removinq from the N and/or C terminal" refers to cleaving or otherwise chemically removing the moiety in ~uestion from the remainder of the peptide.
The term "unaer suitable conditions" means carrying out the method in the presence of the appropriate chemical~, buffers, temperature, for an appropriate time.
Such conditions will be known by tho~e skilled in the art.
In an alternative embodiment related to the third aspect the invention provides a method of activating a lytically inactive peptide, wherein saia inactive peptide com~rises an amphipathic a-hsl;Y of ~ufficient length and character to allow the pe~tide to function lytically and (a) a cleavable moiety located amino terminal to said a-hel;Y, wherein said moiety inactivates lytic function of said re~tide and wherein removal of said moiety results in a lytically active ~eptide, and/or (b) a negatively charged cA-hoYy terminal residue wherein the negative charge may be removed by blocking with a suitable group which results in lytic activity said method com~rising, under suitable conditions, treating said peptide with (c) a cleaving agent to remove said moiety and/or (d) a bloc~; ng agent to remove the negative charge of said residue such that said pe~tide becomes lytically active.
The cleaving agent may be any suitable agent to remove the cleavable moiety. Where the cleavable moiety is an amino acid sequence the cleavin~ agent may be an appro~riate enzyme.
The block; n~ agent may be any agent suitable for blocking the negative charge. In the case where the peptide comprises a cysteine residue, for example, the blocking agent may be another am~hipathic a-helical peptide which co~tA; n~ a cysteine residue and is CArAhl e of forming a dimer. Other suitable block;ns agents may be the suitable groups described above.
The term "under suitable conditions" in relation to the cleaving agent and/or bloc~ agent means treating the pe~tide under a~ro~riate reaction conditions and for an approrriate time such that the cleaving agent and/or blocking agent can ~erform their desired functions. Such conditions will be determined by the nature of the ~eptide~
and the agents used and will be well known to those skilled in the art.
In another alternative embodiment related to the third aspect the invention provides a method of inactivating a lytically active peptide wherein said peptide com~ri~es an amphipathie a-helix of ~ufficient length and eharacter to allow the pe~tide to function lytieally, and/or a non-negatively eharged earboxy terminal residue, and where said ~e~tide is optionally in the form of a dimer eom~rising two a-helice~, said method compri~ing, under ~uitable eonditions, (a) l;nking a eleavable moiety amino terminal to ~aid a-helix and/or (b) creating a negative charge on said carboxy terminus.
Preferably the cleavable moiety is an amino acid presequence which reduee~ the overall ~ositive charge of the peptide.
According to a fourth aspect the invention provides a ~harmaceutieal composition, com~riaing a ~e~tide of the invention, either alone or cou~led to a carrier molecule, together with a pharmaceutically-acceptable carrier. Optionally the composition may also comprise an antibiotic or a toxic anion, sueh as fluoride ion, or an anti-tumour, antibacterial, anti-viral or antiparasite agent.
Melittin i~ known to be able to inhibit the replication of human imm~noA~ficiency virus (International Patent Publieation No. W0 91/08753), and it is contemplated that the pe~tide~ of the invention will be u~eful for thi~
purpo~e, a~ well as in the treatment of caneer.
~eeording to a fifth embodiment, the invention pro~ides a method of treatment or alleviation of cancer or of a manife~tation of infection with human immunodeficiency virus, comprising the step of ~Amin;stering an effective amount of a peptide of the invention, either alone or coupled to a carrier molecule, to a ~ubject in need of such treatment.
According to a sixth embodiment, the invention provide~ a method of treatment of a microbial infection, W097t33908 PCT/AU97/~1 com~risin~ the step of administering an effective amount of a ~e~tide of the invention to a subject in need of such treatment, said dimer com~risin~ an amphi~athic helical sequence of the invention and a cecro~in or a cecro~in-like ~eptide.
According to an seventh embodiment, the invention provides a biosensor, com~rising a first monomeric form of a pe~tide of the invention linked to an antigen and a second monomeric form of a peptide of the invention linked or adsorbed to a solid su~ort or to a membrane, a receptor, or a bound antibody or Fab, Fv, scFv or other antigen-b;n~;ng fragment or analogue thereof, such that an analyte to be detected induces dimerisation of the monomers.
Dimerisation may be detected by membrane lysis or by ion-c~n~l format~on. In the latter ca~e, the dimeric form of the molecule constitutes an voltage-gated ion ch~nnel, 80 that dimerisation results in current flow.
Preferably dimerisation is effected via formation of a disul~hide bond, 80 that the biosensor of this as~ect of the invention pro~ides a redox switch.

Detailed Description of the Invention The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the fi$ures, in which:
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 10-30 minutes and analysed by light scattering and propidium iodide penetration, indicated by fluorescence.
Figure lA show~ flow cytometric analysis of untreated control cells, with side scatter (vertical axis) plotted against forward scatter (horizontal axis), Figure lB shows the viability of control cells, a~ mea~ured using exclu~ion of propidium iodide; cell count (vertical axis) is plotted ~ n~t propidium iodide fluorescence (horizontal axis);
Figure 2 ~hows the effect of intact melittin on cells;
Figure 3 shows the effect of ~e~tide 29;
Figure 4 shows the effects of ~eptide 01B;
Figure 5 shows the effects of ~e~tide 10;
Figure 6 shows the effects of ~eptide 34;
In each of Figures 2 to 6, Figure A 8how8 flow cytometric analysis, Figure B shows cell viability;
Fiqure 7 shows the effects of ~eptide APll;
Figure 8 shows the effect of melittin on cell membrane ~otential, as measured by formation of ion cl~nnel#~ indicated u~ing the dye DiSc3. Figure 8A shows control cells, Figure 8B shows melittin-treated cells.
Figure 9 shows diagrammatic re~resentations of immunotoxin molecules constructea according to the invention.
Figure 9a depicts an immunotoxin in which the inactivating leader ~?eptide i8 joined by a proteolytic cleavage sequence (X) to a cytolytic peptide joined to a scFv shown here as VH-~L. One exam~le of the strategy for the genetic construction of immunotoxin molecules is shown in figure 10.
Figure 9b depicts an immunotoxin in which She chemically or ~enetically conjugated Fab comprising one heavy chain fused to a proteolytically sen~itive linker se~uence (X) fused to a cytolytic pe~tide and a second light chain molecule fused to another proteolytically sensitive linker se~uence ~X) fused to another cytolytic pe~tide. The genetic conjugation is in the description of figure 10.
Figures 9c and 9d depict reverse orientation forms of 9a and 9~.

W097/33~8 PCT/AU97/001 Figure 10 shows the s~ecific oligonucleotide~
u~ed to construct an scFv ex~ression cassette for the NcoI-Eco~I restriction ~ites of ~GC. 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 techni~ue~ that are ~ublished ~Coia G., Hudson, P.J. and ~illey G.G. (1996) Journal of Immunological Methods. 192, 13-23).
Following our study of lytic peptides suitable for the construction of immunotoxins (Werkmei~ter et al, Biochim. Biophys. Acta., 1993 1157 50-54), we ~ynthe~ised a peptide consisting of the first 20 residues of melittin with the addition of a C-terminal cysteine amide. The intention was to cou~le thi~ ~eptide to a monoclonal antibody (Mab) through the cysteine thiol group.
Published reports suggest that the 21 residue peptide could not be lytic in it~ own right, but might assume this activity on delivery to the cell membrane by an Mab (Schroder et al, o~.cit. ) . Surprisingly, the peptide waR found to have high lytic activity, which we have now found was the result of the formation of a dimer, formed from the oxidation of the cy~teine thiol.
Native melittin, free of phospholi~ase, was purchased from Fluka Chemie AG, Switzerland and used without further treatment. Synthetic melittin was obtained from Au~pep, Australia.

Example 1 Synthesis of Peptides Native melittin, free of phospholipase, was purchased from Fluka Chemie AG, Switzerland and used without further treatment. Synthetic melittin was ob~;ne~
from Auspep (Australia).
Synthetic amphipathic peptide~ were de~igned according to the principles set out in the review by Kaiser and Kezdy (Proc. Natl. Acad. Sci. ~SA., 1983 80 1137-1143).

Ac represent~ acetyl and X represents norleucine.
Norleucine was inserted into peptides 10 and 34.
The peptide which we have designated OIB, of sequence S H-G I G A V ~ R V ~ T T G L P A L I S W I C -N~2 was synthesised on an Ap~lied Biosystems 430A Pe~tide Synthesizer, using the FastMoc strategy. RINK resin (Rink:
Tetrahedron Letters, 1987 728 3787) was used as the substrate for the assembly of this peptide, yielding the ~e~tide as the amide on cleavage with trifluoroacetic acid.
The cysteine side chain was ~rotected by trityl, serine and threon;ne by t-butyl and lysine by t-butyloxycarbonyl groups. The ~e~tide was purified by reverse-pha~e HPLC
using a VYDAC C18 column. Sel?aration was achieved by using a 20 minute gradient from 5 to 60% acetonitrite. The integrity of the ~eptides was established by amino acid analysis and electrospray mass s~ectrometry. The ~eptide oxidised when dissolved in dimethyl sul~ho~;Ae in the presence of air to yield the disulphide dimer. Monomeric pe~tide (OlD) was prel?ared by alkylating with iodoacetic acid in dimethylformamide. Monomeric peptide 63 was prepared as described above using ACI!~.

The lysine brAnch~ dimer, ~eptide 30, was synthesised by the technique described above, but Fmoc~ys(Fmoc)OH was used at the C-terminal. This yielded a crude product which consisted of a mixture of deletion peptides along with the de~ired peptide which was purified by C1B re~erse 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.
The acetylated peptides, 01C, 42B, 44B and 46B

CA 02248782 l998-09-ll WO 97/33908 PCT/AUg7/00160 were obt~;ne~ by treating the parent ~e~tides with an excess of acetic anhydride in dimethylformamide cont~;n;ng a trace of triethylamine.
Other ~eptides, whose ~e~n~e~ are given below, were synthesised e~entially as de~cribed above and likewise, where a~pro~riate, dimerised by oxidation of the thiol of the cysteine residues.

GIG A V L R V L T T GLP A LISWIC-NH2 (Peptlde OlB) AC-G I G A V L lC V L T T G LP A L I S W I C -Nl12 ~Peptlde OlC) 0 Ac GIGAVLRVL T T GLPAL I S W I C -~8C~2COO~ 2 (Peptlde OlD) AC-X LQALLSLLQSL L SLLLQFLRRRRQQ-NH2(Peptlde 10) CGIGAVL R V LTTGLPALISWI-Na2(Peptlde 29) 2tGIGAVL R V LTTGLP A LISW)R-~I2(Peptlde 30) X LQSLVS L V QSLVSLV L QFLRSRRNN-~2(Peptlde 34) GLG A LV R LVTSGVPLVLSWLC-NI~2(Peptlde 56) GIG A V LRVL T T G LP A LISWIC-~s~ ,) -~,(Peptlde 63) LLQSLVSLVQSLVSLVLQPLC-NU2(Peptlde 41) LLQSLV R L VQSLVPLVLQFLC-N~2(Peptlde 42) Ac-LLQSLVKLVQSLVPLVLQF L C~2(Peptlde 42E~) Ac LLQSLV}CLVQSLVSLV L QFLC-N~2(Peptlde 44) AC-L L QSLVXLVQSLVSLVLQFLC-N~2(Peptlde 44B) AC
LLQSLVRLVQSLVP L VLQFLC-NEI2(Peptlde 46) LLQSLV~CLVQSLVPLV L QF L C(6C~I2COOEI~-N82 (Peptlde 42C) Ac-LLQSLVR L V QS L V P L V LQF L C-~2 (Peptlde 46}3) A V R G I G A V L R V LT T G LP A LISWIC-~H2 (Peptlde 50) AC-A V R G I G A V LI~VLTTGLP A LISWIC-N~12 (Peptlde SOA) SSLG I R AVL R V LTTGLPALISW I A -~2(Peptlde APll) SSLGIGAVLKVLTTGLPALISWIA-N~2(Peptlde AP 13) In these peptides, ~heny~ n;ne can be substituted for tryptophan without 1088 of activity.

Example 2 Haemolytic Effects The haemolytic effect of the peptides of Example 1 was compared with that of melittin.

W097/33~8 PCT/AU97/~1 Peptides were ai~sol~ed in dimethyl ~ p~ox;~e (DMSO) at 5 mg/ml and ~erially titrated by two-fold ailutions in phosphate buffered r~ ;ne (PBS). The final CQ~C~ ations in the 96-well ~-bottomed microtitre plates S (Nunc, Denmark) ~e~ from 200~g/ml to 0.7~g/ml. 0.6%
suspen~ion of washed human red blood cells (100~1) were added for 1 hour. Plates were centrifuged at 150 X g for 5 min, and 100~1 aliquots were transferred to a 96-well polyvinyl chloride ~late (Dynatech ~aboratories, Al~Y~n~ria, VA). Haemolysis was aQsessed by measurement of optical density at 405nm with an automatic EAR 400 SF ~LISA
plate reader (SLT Lab instruments, Groedig/Salzburg, Austria). The percentage of haemolysis was calculated by comparison with absorbances from a buffer blank ("no lysis"
control) and a sample treated with 0.1% Triton X-100 (~maximum lysis" control). ~ytic acti~ity could be calculated from the linear ~ortion of the haemolytic titration curve for each peptide where one lytic unit was defined as the concentration of peptide re~uired to produce a given percentage of haemolysis. The relative acti~ities of peptides were assessed directly from the linear portion of the titration curve~.
The results are summarised in Table 1.

CA 02248782 l998-09-ll W 0 97~3908 PCT/AU97/00160 Table 1 % ~aemolysis Induced by Pe~tides, as compared to ~elittin (100%) Final Co~ nf,~tion of Peptlde (~g/ml) Peptide 200 ~00 50 25 12 6 3 1.5 0.7 5 Mellttln 100 100 100 100 100 65 51 14 4 OlB 100 100 96 58 29 14 9 4 2 OlB 97 100 96 83 41 17 12 9 9 OlC 1 0 1 0 0 0 0 0 0 OlD 1 0 1 1 0 0 0 0 0 APll 100 97 81 34 10 5 3 3 4 Pep ide 2 : C-terminal c~steine Peptide 30 : dimer cross-linked ~ria lysine residues R/A : reduced and alkylated WO 97/33908 PCT/AUg7/00160 Table 1 summarise~ the haemolyic effects of all ~e~tides after 1 hour at various cQn~ent~ations ranging from 200 ~g/ml to 0.7 ~g/ml. Pe~tide 10 i8 based on the sequence ~ubl;Rhe~ by Degrado et al ((1981) J.Amer.Chem.Soc.103:884-890; the variations being the re~lacement of the N-terminal leucine with N-acetyl norleucine, trypto~han 19 with ~heny~ n;n~ and lysine 21 with arginine . Des~ite ret~; n; n~ only 31% sequence identity with melittin, peptide 10 has a ~imilar lytic activity to melittin (Table 1). Likewi~e peptide 34, which ha~ no sequence identity with melittin has only a slightly re~ce~ activity, ~omewhere around a 4-fold reduction.
Pe~tide OlB, the truncated (C-terminus-deficient) analogue of melittin was found to be highly active causing ~ignificant haemolysis at cG~c_~Lrations as low as 12 ~g/ml. The N-acetylated form of this ~eptide (~e~tide OlC) and the monomer (~eptide OlD) were both com~letely inactive even at the highest co~c~nt~ations of 200 ~g/ml.
Dimeri~ation of this truncated pe~tide either by an N-terminus cysteine (~eptide 29) or by a C-terminus lysine (peptide 30) also re~ulted in highly lytic ~eptides, slightly less toxic than pe~tide OlB (Table 1).
Peptide 41, the truncated (C-terminus-deficient) analogue of the lytic ~e~tide 34, wa~ completely inactive even at the highest concentration. Substitution of lysine and ~roline into this sequence at ~ositions 7 and 14 respectively (peptide 42), resulted in significant lytic activity, albeit still around 4-fold less than pe~tide 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 sub~titution of proline at position 14) produced a highly lytic peptide (peptide 46, Table 1) which was e~ually if not more active than peptide 42. In all these studies with amino acid substitutions, the haemolytic activity of the resulting CA 02248782 l998-09-ll ~e~tides required a free unacetylated N-terminus (pe~tides 42C, OlC, 42B, 44B and 46B.
We al~o examined the activity of monomeric ~e~tides whose sequence was hA~ on that of melittin. Pe~t~des 10 and 34, each of which has N-terminal norleucine, leucine replacing lysine7 and serine re~lacing ~rolinel4, both have haemolytic activity comparable to that of melittin.
However, neither of these ~e~tides is active in the dimeric form. Thus, contrary to the results of Blondell and Houghton (o~.cit. ) it a~ears that a residue corres~onA;ng to lysine7, is not required for activity of the monomeric ~eptide; nonetheless, this residue confers maximNm activity on the dimer, as does a proline corres~onA; n~ to ~rolinel4.
In Peptide 29, cro88-l; nk; ng is via a cysteine added at the C-terminal of the melittin sequence, 80 that the lysine Is at position 8 and proline is at ~osition 15 in the ~equence of this ~e~tide; good activity was observed, ~o it is clearly essential only that a lysine and a proline occupy these relative ~ositions in the sequence.

Example 3 Mea~urement of CytolYtic Effect by Flow Cytometry To assay the effects of cytolytic pe~tides on cells, we have utilised flow cytometry as described by Weston et al (Cytometry, 1994 15 141-147), with some modifications. Flow cytometry uses analysis of the scattering of laser light from cells moving in a fluid stream to give information about the state of the cell~.
Light ~cattered in the direction of the laser beam (forward scatter) is measure of cell size. Light scattered at right angles to the beam (side scatter) is an indication of gr~n~lA~ity within the cell, and hence of cell m~hrane integrity. In addition, fluorescent molecules can be added to the cells, and their specific fluorescence when excited by the la~er 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, dep~n~; ng on the specific fluorescent dye used.
Weston et al showed that cells change their forward and side light scattering ~ro~erties when acted u~on by melittin. We have mea~ured these changes, and have also incorporated two additional ~arameter~. The first is cell viability measured by the exclusion of the dye pro~idium iodide. Cells with intact membranes do not take u~ this dye, and do not become fluorescently stA;neA.
Cells with broken membranes take up the dye, which binds to their internal nucleic acids and becomes brightly fluore~cent. Cell viability can therefore be measured at the same time as light scattering, and i~ indicated by the pro~ortion of cells that are not st~; n~ by propidium iodide. A~ an index of ~e~tide cytolysis, forward light scatter was measured and ~lotted again~t cell membrane integrity which was assessed by exclusion of the dye ~ro~idium iodide (Shapiro (1994) Practical Flow Cytometry, Third Edition, Wiley Lis~ New York). CEM T cell lymphoma cells (250~1) were used for the cytolytic a~ay, at a cQnc n~ation of 106 cells/ml in PBS. Cells were incubated at room temperature in the presence of peptides and 4~g/ml propidium iodide (Sigma Chemical Com~any). The 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 t 600nm.
Fluorimetric meaaurement of cell membrane potential was also determined. Some carbocyanine dyes dissolve in cell membranes and give a fluorescent signal which is depen~ent on the orientation of the dye molecules within the cell membranes. (Cohen & Salzberg (1978) Rev.
Physiol. Pharmacol. 83:35-88). This orientation is dependent on the voltage difference across the cell W O 97~3908 PCT/AU97/00160 membrane and therefore the ;n~en~ity of the fluorescent ~ignal is ~ro~ortional to the voltage drop. Cells that are effected by cytolytic ~e~tides develo~ ion c~Ann~l~ that decrease the cell membrane ~otential, resulting in a rapid decrea~e in dye fluorescence. We have used the carbocyanine dye, DiSC3 (Ash et al (1978) Biochim. Biophys.
Acta 1030:1-10) [3,3'-diisopropyl~h;~;carbocyanine iodide]
(Molecular Probes, Eugene, Oregon), to measure cell membrane potential and indicate the formation of ion ch~nn~ls after addition of pe~tides (100~g/ml) for 5 minutes. For membrane ~otential measurements cells, in PBS, were pre-loaded with 0.05~M DiSc3 in normal saline for 15 min. at room temperature. Flow cytometry was carried out a~ above with illumination at 488nm for light scatter and at 633nm for excitation of DiSC3 fluorescence.
Fluore~cence of the DiSC3 was measured at greater than 633nm. ~ec~ e the fluore~en~e emi~sion spectra of ~ro~idium iodide and DiSC3 overla~ (Schapiro (1994) o~
cit), the two dye~ could not be used simult~n~o~-~ly.
Re~ult~ with untreated control cells are ~hown in Figure 1, and results with intact melittin are shown in Figure 2. Figures 3, 4, 5, 6 and 7 respectively show results with pe~tide 29, peptide 01B, ~eptide 10, peptide 34 and ~eptide APll.
The dots on the diagram in A for each figure are measurements on individual cells, and their density indicates the number of cells in each area of the diagram.
Figure lA shows a normal cell population. It can be seen that the measurements of mo~t cell~ 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 lB shows normal cells that are not fluorescent, indicated by the histogram peak against the left axiq of the graph. Dead or damaged cells have fluoreseence that iB greater than that observed for the normal cells, ;nA;e~ted by a shift in the histogram toward the right of the diagram.
It is evident from the figures that the different peptides have differential effeets on light scattering and eell viability.

Example 4 Effeet of Cytolytic Peptides on Cell Membrane Potential Carbocyanine dyes are fluore~cent probes whieh dissolve in eell membranes and give a fluorescent signal which is de~nA~nt on the orientation of the dye molecules within the cell membranes. This orientation is dependent on the voltage difference acro~s the cell membrane, and therefore the ;ntenaity of the fluore~eent signal is proportional to the voltage drop. Cells that have been affected by cytolytie peptides develop ion chAnnDl~ that deerea~e the cell membrane potential, resulting in a shift of the fluorescence inten~ity histogram towards the left of the diagram. We have used the carbocyanine dye DiSC3 (3,3~-diisol?ropyl~h; AA; earbocyanine iodide; Moleeular Probes, Eugene, Oregon) to measure eell membrane potential and indicate the formation of ion ch~nnsl~. This procedure i~ a very ~ensitive means of demon~trating the effects of eytolytic peptides. CEM T eell lymphoma cells were ;nc~lh~ted with 5 mM DISC3 in normal ~aline for 15 minutes at room tem~erature, and followed over a period of O to 10 minutes.
Results with untreated control cells and with eells treated with melittin for S min are shown in Figure 8A and Figure 8B respectively. It is evident that almost the entire population of eells is rendered permeable by melittin.

Bxam~le 5 Construction of Pe~tides CAr~hle of Lytic Activity Peptides capable of lytic activity in accordance with the second as~ect of the invention may be ~roduced in accordance with the following. While the following relates to dimers it will be a~preciated that monomeric pe~tides may be similarly produced.
Synthe~ The ~e~tide H - G I G A V L R V ~ T T
G ~ P A L I S W I C -NH2 was synthesised and oxidi~ed to produce a disulphide dimer basically as described in ~xample 1.
Peptiden such as OlB, 42 and L L Q S L V R L V Q S ~ V P ~ V L Q F ~ C -N~2 (Peptide 46) may be ~ynthesised as described in Example 1. The inactivating ~resequence may be A~Ae~ during ~eptide synthesis or the relevant nucleic acids encoA;~g it may be included in the construct if the ~e~tide i~ being made by recombinant means. ~t should be noted that phenyl ~A l An; ne may be substituted for tryptophan with only minimal 1088 of lytic activity.
The len~th of the bridge between the two peptide chA;nQ is relatively flexible and the disul~hide can be replaced by either a - CH2 S (CH2)X S CH2- (where x i~ 1-4) crosslink or a lysine or ~mall lengths of pe~tide or other chemical moiety.
Deactivation:
We have discovered that the ~eptides described above, and related sequences, can be rendered inactive by the addition of an appropriate pre-sequence, then reactivated by treatment with an ap~ro~riate enzyme. The presequences are added as required and their sequence being dependent on the enzyme which is plAnne~ to cleave at the appropriate site.
Typical presequence~: (inactivated by) Acetyl-A V Y- (Prostate Specific Antigen or Chymotrypsin) Acetyl- S S G Y S N T - (PSA or Chymotrypsin) CA 02248782 l998-09-ll W097/33~8 PCT/AU97/00160 Acetyl-A ~ E- (St~phyloc~c~- ~ au~eu~ V8 protea~e, Houmard, J. & Drapeau, G. R. (1972), Proc.Natl.Acad.Sci.~SA, 69,3506-3509) Many other prese~n~e~ are possible, only requiring a specific cleavage site which is not ~re~ent in the main peptide chain.

Example 6 Construction of Additional Peptides The following ~eptides are constructed in accordance with Exam~le 1 and tested for their haemolytic activity.
G I G A V L R V L T T G ~ P A ~ I S W I A-NH2 (Peptlde 64A) G I G A V L R V h T T G L P A ~ I S W I N-NH2 (Peptide 64N) G I G A V L R V L T T G L P A ~ I S W I D-NH2 (Peptide 64D) G I G A V L R V L T T G L P A L I S W I H-NH2 (Peptlde 64H) G I G A ~ L R V L T T G ~ P A ~ I S W I I-NH2 (Peptide 64I) G I G A V L ~ V L T T G L P A L I S W I P-NH2 (Peptide 64F) G I G A V L R V ~ T T G L P A L I S W I S-NH2 (Peptlde 64S) G I G A V L R V L T T G L P A L I S W I S-N~2 (Peptide 64M) The major f;n~;n~ of the ~re~ent study de~cribed in the Examples was that the disulphide dimer, peptide OlB, had a lytic activity approAch;n~ that of melittin (on a molar basis), despite the reports that shortened sequences of melittin lose acti~ity, r~Ach;n~ zero on the 108~ of the basic C-terminal sequence. This was further demonstrated by alkylating ~eptide OlB (~e~tide OlD) to give a non-active monomeric peptide. However, if the cynteine thiol was blocked by acetamidomethyl (ACM), a non-charged group, lytic activity was retA;ne~, only marginally reduced compared with melittin. These results sugge~t that the pre~ence of the negative charge on the carboxymethyl group could be res~onsible for the 10~8 of activity and that lytic activity may not be dependent on the formation of a dimer. If the ~osition of the cross-link is placed at the N-terminal, instead of the C-terminal, the activity is reduced but is still ~ignificant (peptide 29).

CA 02248782 l998-09-ll W O 97/33908 PCT/AUg7/00160 The ~equence requirement~ of the~e dimers appear~
different to that of melittin, and the ~e~tide analogues de~cribed earlier, a~ a dimer (~eptide 41) ba~ed on the ~ecluence of one of these analogue~, ~eptide 34, has no ~igni$icant activity. Various ~ub~titution~ indicate that proline 14 and a ba~ic side chain at ~o~ition 7 are e~ential for haemolytic activity of the dimeric ~eptides.
Furthermore acetylation of any of theRe dimeric pe~tide~
reduce~ or de~troys their activity (~eptides OlC, 42B, 44B, 46B and 50A). We have included an aromatic residue at position 19 in all of these peptides becau~e of the reported im~ortant role trypto~han ~lays in the activity of melittin.
We thus have the ~ituation where it ha~ been demon~trated that lysine 7 i~ not important for the lytic activity of melittin and melittin-like pe~tide~, but either ly~ine or arginine a~ears essential for the activity of the dimeric peptide~. ~his i~ reinforced by the observation that acetylation of ~eptide OlB (~eptide OlC) destroys its activity, whereaa acetylation of melittin ha~
no effect (Habermann & Rowallek (1970) Hoppe - Seyler'~ Z.
Physiol. Chem. 351 : 884-890).
A ~imilar ob~ervation is evident with ~roline 14, where it~ inclusion enh~nce~ activity of the dimer, but removal of ~roline from melittin ha~ either no effect or even a positive ~nh~n~ement of activity, peptide~ 10 and 34 (Degrado et al (1983) Biophy~. J. 37: 329-338; Werkmei~ter et al (1993) Biochim. Biophy~. Acta 1157:50-54.
A summary of the~e re~ults would ~ugge~t that a lytic peptide with activity a~pro~ch;ng that of melittin can be produced from an a~proximate 20 residue amphipathic peptide providing the N-terminal amino group remains free and the ~eptide include~ a lysine or arginine residue at position 7, a proline at 14 and probably an aromatic re~idue at 19. A cro~ link at the N-terminal is less effective than one at the C-terminal. Despite these W097/33gO8 PCT/AU97/~1 conclu~ion~ it i~ noted that peptiae OlB i~ the mo~t lytic of the dimeric peptides pro~-~ce~, and that there i~ clearly ~ome other sequence parameter involved, other than tho~e already discu~ed. Nonethele~s, thi~ ~tudy provide~
~ign~ficant information for the generation of a unique cla~ of toxic peptide~ from truncated analogue~ by ~election of certain e~ential reQidue~ and dimeri~ation.

Example 7 Preparation of Recombinent Immunotoxin~
Fiqure 9 depict~ cartoon diagrams of immunotoxin~.
Figure 10 de~cribe~ ~pecific oligonucleotides which were ~ynthesiQed and used to con~truct an scFv (a~
~hown in Fi~ure 9a) as an expre~ion ca~ette in the Ncol-EcoRl site of vector pGC. (Coia et al, J. Immunol Methods, 1996 192 13-23). The ~ynthesised ~rotein, after expre~ion and Qignal ~eptide cleavage, compri~ed N-terminal F~AG
octapeptide fu~ed to the tripeptide AVY ~PSA cleavage ~ite) fu~ed to DEROlB cytolytic peptide fu~ed to GGGG ~pacer fu~ed to an ~cFv. In thi~ example the ~cFv was derived from hybridoma lC3 (glycophorin Qpecific; Coia et al, 1996). The recombinant i unotoxin wa~ purified (cytolytically inactive) until pre~entation in vi~o where activation i~ dependant on cleavage by pro~tate-specific antigen (PSA). It should be recogni~ed that any alternative enzymatic cleavage ~equence could replace AVY, and that any cytolytic peptide described in thi~
~pecification could replace DEROlB and that any cFv can replace lC3.
Figure 9a depict~ an immunotoxin in which an inactivating leader peptide is joined by a proteolytic cleavage sequence (X) to a cytolytic ~eptide joined to an ~cFv (in thi~ ca~e in the orientation VH-V~). The details of the genetic con~tructs are described in Figure 10.
Figure 9b depict~ a chemically or genetically conjugated Fab comprising one heavy chain fuqed to a W097/33908 PCT/AU97/OOlGO

cytolytic ~eptide and a second light chain molecule fused to another ~roteolytically ~en~itive linker se~uence fused to another cytolytic pe~tide. The details of methods of genetic construction will be known to ~erson~ skillea in the art from ~revious de~cri~tions such a~ Dolezal et al, (Immunotechnology, 1995, 1 197-219). ~t should be recognised that the cytolytic peptide~ will be disuphide bonded through cysteine residues and thereby remain inactive until ~roteolytic cleavage occurs at the linker regions.
Figures 9c and d depict reverse orientation forms of Figures 9a and 9b.

Application~ of the Invention These peptides can be used in a wide range of application~ which exploit the ~roperty that the pe~tides ha~e little or no biological activity either in the monomeric ~tate and/or when the C-terminal residue is negatively charged, but are highly lytic on dimerisation and/or blockage of the negative charge.

1. BioseD~ors In this ap~lication it is envisaged that one lytically inactive monomeric form of the peptide i8 attAc~A to an antigen and a second i~ att~AcheA or adsorbed to a ~olid su~ort or to a membrane, a receptor, a bound antibody or a Fab fragment, the measurement being a~ a re~ult of membrane lysis or by ion-chAn~el formation. In the latter case the dimeric form of the molecule constitutes a voltage-gated ion chAnnel, ~o that dimerisation results in current flow, effectively pro~iding a redox switch if dimerisation is via a disulphide bond.

W097/33908 PCT/AU97/~1 2. ImmDn~
In thi~ application the peptide is attAcheA, by means of a suitable cro~-l; nk; ng agent, either as a monomer or as a dimer, to an antibody or Fab fragment, Fv fragment or ScFv fragment or other antigen-specific fragment thereof, which is specific for a target cell.
Preferably the antibody is directed to a target antigen which does not mediate internalization, more preferably a carcinoma-specific antigen. The synthetic peptide~
described herein are designed for ease of 1; nk; ng to antiboaies or fragments thereof. It is contemplated that they will be useful as replacements for toxic peptide~ such as Ricin A chain. Representative constructs are depicted in Figure 9.
In an alternative approach the peptide of the invention i~ expressea either as a monomer or as a -heterodimer by recombin~nt DNA technology, together with the Fab, Fv or ScFv fragment of an antibody. The immunotoxin can be utilised as the monomer, or can be dimerised prior to utili~ation.
In another alternative, the monomeric peptide is crosslinked to the immunoglobulin or Fab fragment, with the same or different monomer attAch~ thereto. One monomer chain is attAche~ to the protein and the second free peptide crosslinked to it, preferably via the formation of a disulphide.
A third a}ternative 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.
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 W097/33908 PCT/AU97/~1 active molecule. Other method~ will readily occur to the person skilled in the art.
For targeting tumour cell~, such as prostate cAncer cells, a peptide of the invention can be expressed with one or more tyrosine residues incorporated direct}y adjacent to or near to the ~eptide sequence, which are then cleaved by the target cell. While ~uch clea~age may not be essential for acti~ity, activity could be increased in this way. We have found that ~roQtate-specific antigen has protease activity which induces specific cleavage at tyr-tyr (see PCT/A~95/00536 entitled "As~ay for the Detection of proteases").

3. Bacter;c; ~e~/~n~; h- otic~
~sing two different designed se~lence~ for each helical segment it is possible to ~roduce a dimer which will ly~e bacterial cells but not mammalian cells. For example the amphipathic helical sequence:

~-G I G A V L X V L T T G I- P A L I S W I C ~ 2 may be coupled to a suitable segment from a class of peptides known a~ cecropins. Some cytolytically- and antibacterially-active cecropin A - melittin hybrid peptides are known (D;~-ch;rica et al, ~ur. J. Biochem., 1994 224 257-263; Fsrns~n~lez et al, Biopolymers, 1994 34 1251-1258). Through the formation of a disulphide, such a segment may suitably be:

R W R L F R R I E X V G Q C -N~2.

Alternati~ely, to aid in the formation of the disulphide, the cysteine residue may be replaced by a penicillamine residue in one of these peptide~.
These peptide sequences are only examples, and potentially any amphipathic helical sequence could be W097133~8 PCTIAU97100160 ~ubstituted for the f~ir~t peptide and likewi~e several alternative ~eq~nce~ derived from the cecropinR could be sub~tituted for the Qecond peptide.

4. The~ l;c Treatment of Disease The peptide~ of the second embodiment of the invention are tho~ht to be particularly u~eful in the treatment of di~ea~e~ where it is nece~sary to ~electively de~troy cell~ ~uch as in cancer therapy or in other therapeutic ~ituation~ where it is desirable to deRtroy cell~, preferably ~electively. The~e peptide~ can be u~ed in application~ which exploit the ~roperty that the peptides have little or no biological activity until activated by an appropriate enzyme.
In cancer treatment the peptide is de~igned to be released from it~ inactive form in the pre~ence of a tumour specific protea~e, ~uch as PSA. In an alternative approach an a~propriate enzyme can be delivered to the ~ite of a tumour by an antibody prior to treatment with the ~eptide.
In a second alternative the pre-toxin is att~c~e~, by means of a Ruitable cross-l;nk;ng agent, either as a monomer or as a dimer to an antibody or Fab fragment. Alternatively the monomeric peptide is expre~sed with the Fab fragment.
It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purpose~ of clarity and under~tAn~;ng, variou~ modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this apecification.

Claims (40)

- 37 -

1. A peptide with lytic activity, having an amphipathic .alpha.-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.

2. A peptide according to Claim 1 which the amphiphathic .alpha.-helix is derived from a naturally-occurring cytopathic amphipathic peptide, selected from the group consisting of melittin; other insect venoms; wasp, ant, and scorpion venoms; magainins, bombolitins, and mastoparans;
and other known cytopathic amphipathic peptides.

3. A peptide according to Claim 1 comprising two .alpha.-helices which are the same.

4. A peptide according to Claim 1 comprising two .alpha.-helices which are different.

5. A peptide according to any one of Claims 1 to 4 comprising up to 21 amino acids, in which the .alpha.-helix has at least 45% amino acid homology to melittin.

6. A peptide according to Claim 1, having hydrophilic amino acids at positions 3, 4, 7, 10, 11, 14 and 18, of which at least two are lysine and/or arginine;
and hydrophobic amino acids at positions 1, 2, 5, 6, 8, 9, 12, 13, 15, 16, 17, 19 and 20.

7. A peptide according to Claim 6 in which the amino acid at position 7 is lysine or arginine.

8. A peptide according to Claim 6 or Claim 7 in which the amino acid at position 14 is proline or hydroxyproline.

9. A non-naturally occurring peptide with lytic activity, having an amphipathic .alpha.-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.

10. A peptide according to Claim 9 in which a cysteine residue is present at either the N-terminal or the C-terminal end of the .alpha.-helix.

11. A peptide according to Claim 10 in which the C-terminal residue is a cysteine residue which has its thiol group blocked.

12. A peptide according to Claim 1, Claim 3 or Claim 4, comprising two amphipathic .alpha.-helices which are not themselves lytic, linked to form a dimer which has lytic activity.

13. A peptide according to Claim 12 in which the components of the dimer are linked via a disulphide bond.

14. A peptide according to Claim 13 in which the disulphide bond is 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 -CH2S(CH2)nSCH2-, where n is 1 to 4.

15. A peptide according to any one of claims 12 to 14 in which the amphipathic .alpha.-helix is derived from a naturally-occurring cytopathic amphipathic peptide, selected from the group consisting of melittin; other insect venoms; wasp, ant, and scorpion venoms; magainins, bombolitins, and mastoparans; and other known cytopathic amphipathic peptides.

16. A peptide according to Claim 12 which is a cytotoxic dimer of an amphipathic peptide, in which the amphipathic peptide has the general formula:
.OMEGA. .OMEGA. X X .OMEGA. .OMEGA. X .OMEGA. .OMEGA. X X .OMEGA. .OMEGA. (X, P or hypro) .OMEGA. .OMEGA. .OMEGA. X .OMEGA. .OMEGA. (C or Z) ' or (C or Z) .OMEGA. .OMEGA. X X .OMEGA. .OMEGA. X .OMEGA. .OMEGA. X X .OMEGA. .OMEGA. P .OMEGA. .OMEGA. .OMEGA. X .OMEGA. .OMEGA.;

and wherein the symbols have the following meanings:
.OMEGA. = glycine; a hydrophobic amino acid including but not limited to alanine, isoleucine, leucine, valine, and norleucine; or ~;

.SIGMA. = asparagine, glutamine, serine or threonine;
.PHI. = phenylanine, tryptophan, tyrosine, a non-naturally occurring aromatic amino acid, or a synthetic aromatic residue; and X represents a sequence of hydrophilic amino acids, at least one of which is a basic amino acid.
and wherein C or Z may be at either end of the peptide but not at both ends.

17. A peptide according to Claim 17 in which two or more of the hydrophilic amino acids are basic amino acids.

18. A peptide according to Claim 16 or Claim 17 in which the amphipathic peptide has the general formula .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. (K or R) .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. P .OMEGA. .OMEGA. .OMEGA. n .SIGMA. .PHI. .OMEGA. (C
or Z) or .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. (K or R) .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. P .OMEGA. .OMEGA. .OMEGA. .SIGMA. .PHI. .OMEGA. (Cor Z) or (C or Z) .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. (K or R) .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. P .OMEGA. .OMEGA. .OMEGA.
.SIGMA. .PHI. .OMEGA.
or .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. (K or R) .OMEGA. .OMEGA. .SIGMA. .SIGMA. .OMEGA. .OMEGA. P .OMEGA. .OMEGA. .OMEGA. .SIGMA. .PHI. .OMEGA.
and wherein the symbols have the following meanings:
R = is arginine R = lysine hypro = hydroxyproline P = proline C = cysteine Z = is an amino acid which is not negatively charged, or is either a cross-linking agent, or a blocking group.

19. A peptide according to Claim 9 selected from the group consisting of G I G A V L K V L T T G L P A L I S W I C -NH2 (Peptide 01B) C G I G A V K L V L T T G L P A L I S W I -NH2 (Peptide 29) 2(G I G A V L K V L T T G L P A L I S W)R -NH2 (Peptide 30) X L Q S L V S L V Q S L V S L V L W F L R S R K N N -NH2 (Peptide 34) L L Q S L V K L V Q S L V P L V L Q F L C -NH2 (Peptide 42) L L Q S L V K L V Q S L V S L V L Q F L C -NH2 (Peptide 44) G L G A L V K L V T S G V P L V L S W L C -NH2 (Peptide 56) A V R G I G A V L K V L T T G L P A L I S W I C -NH2 (Peptide 50) Ac A V R G I G A V L K V L T T G L P A L I S W I C -NH2 (Peptide 50A) G I G A V L K V L T T G L P A L I S W I M-NH2 (Peptide 64M) S S L G I K A V L K V L T T G L P A L I S W I A -NH2 (Peptide AP11) S S L G I G A V L K V L T T G L P A L I S W I A -NH2 (Peptide AP13) where -NH2 indicates the C-terminal amide group; in which the peptide may optionally be amidated, and/or acylated at the N terminal.

20. A peptide according to Claim 19, in which the helical dimer is G I G A V L K V L T T G L P A L I S W I C
G I G A V L K V L T T G L P A L I S W I C

21. A peptide which is capable of lytic activity, comprising an amphipathic .alpha.-helix of sufficient length and character to allow the peptide to function lytically, and (a) a cleavable moiety located amino terminal to said .alpha.-helix of sufficient length and character to allow the peptide to function lytically, and (b) a negatively-charged carboxy terminal residue of said peptide, wherein the negative charge prevents or retards lytic function of said peptide and wherein removal of said charge by blocking with a suitable group results in lytic activity.

22. A peptide according to Claim 21, in which the cleavable moiety is pre-sequence Acetyl-AVY-, Acetyl-SSGYSNT- or Acetyl-AVE-.

23. A peptide according to Claim 21 or Claim 22 in which the cleavable sequence is selectively removable by enzymatic means.

24. A composition comprising a peptide according to any one of Claims 1 to 23, together with a pharmaceutically-acceptable carrier.

25. A peptide according to any one of claims 1 to 23 linked to a carrier molecule.

26. A carrier-linked peptide according to Claim 25 in which the carrier molecule is a targeting agent.

27. A carrier-linked peptide according to Claim 25 or Claim 26 in which the carrier molecule is an antibody, an antigen-specific fragment or analogue of an antibody, epidermal growth factor or transforming growth factor-.alpha..

28. A carrier-linked peptide according to claim 27 in which the antibody, or fragment or analogue thereof, is specific for a cell-surface antigen.

29. A carrier-linked peptide according to Claim 28 in which the antigen is a cancer-specific antigen.

30. A carrier-linked peptide according to claim 29 in which the cancer is a solid tumour.

31. A composition comprising a carrier-linked peptide according to any one of claims 25 to 29, together with a pharmaceutically-acceptable carrier.

32. A method of modulating the lytic activity of a peptide, said peptide comprising an amphipathic .alpha.-helix of sufficient length and character to allow the peptide to function lytically, and optionally comprising additional amino acids, said method comprising, under suitable conditions, (a) increasing the positive charge of said peptide by adding to the N and/or C terminal one or more moieties which increase the positive charge of the peptide, or (b) decreasing the positive charge of said peptide by removing from the N and/or C terminal one or more moieties which decrease the positive charge of the peptide wherein 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.

33. A method of activating a lytically inactive peptide, wherein said inactive peptide comprises an amphipathic .alpha.-helix of sufficient length and character to allow the peptide to function lytically and a) a cleavable moiety located amino terminal to said .alpha.-helix, wherein said moiety inactivates lytic function of said peptide and wherein removal of said moiety results in a lytically active peptide, and/or b) a negatively charged carboxy terminal residue wherein the negative charge may be removed by blocking with a suitable group which results in lytic activity said method comprising, under suitable conditions, treating said peptide with c) a cleaving agent to remove said moiety and/or d) a blocking agent to remove the negative charge of said residue such that said peptide becomes lytically active.

34. A method of inactivating a lytically active peptide wherein said peptide comprises an amphipathic .alpha.-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 .alpha.-helices, said method comprising, under suitable conditions, a) linking a cleavable moiety amino terminal to said .alpha.-helix and/or b) creating a negative charge on said carboxy terminus.

35. A method according to claim 31 in which the cleavable moiety is an amino acid presequence which reduces the overall positive charge of the peptide.

36. A composition according to Claim 24 or Claim 30, further comprising one or more antibiotics, toxic anions, anti-tumour agents, antibacterial agents, antiviral agents or antiparasitic agents.

37. 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 according to any one of Claims 1 to 23, either alone or coupled to a carrier molecule, to a subject in need of such treatment.

38. A method of treatment of a microbial infection, comprising the step of administering an effective amount of a peptide according to any one of Claims 1 to 23 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.

39. A biosensor, comprising a first monomeric form of a peptide according to any one of Claims 1 to 23 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 receptor, or a bound antibody or Fab, Fv, scFv or other antigen-binding fragment or analogue thereof, such that an analyte to be detected induces dimerisation of the monomers.

40. A peptide, carrier-linked peptide, composition, method or biosensor, substantially as hereinbefore described with reference to the examples and drawings.