CA2174943A1 - A new ribosome-inactivating protein isolated from the plant bryonia dioica - Google Patents

Abstract

The present invention discloses a new ribosome-inactivating protein, bryodin 2, isolated from the plamt Bryonia dioica. This ribosome-inactivating protein (RIP) is a type I RIP having a single polypeptide chain and no cellular receptor domain. Like many type I RIPs, bryodin 2 has a molecular weight of about 27,000 daltons and pI of 9.5. Bryodin 2 differs from previously identified ribosome-inactivating protein in its amino acid composition, amino acid sequence, and toxicity in vitro and in vivo. Bryodin 2 is useful, as are other type I ribosome-inactivating proteins, as am abortifacient, immunomodulator, anti-tumor or anti-viral agent. Compositions comprising bryodin 2 as am immunoconjugate or fusion molecule are particularly useful to kill cells of a target population.

Description

WO 95111977 . 2 1 7 4 q ~ ~ PCT/[JS94/12.-i82 A NEW RIBOSOME-INACTIVATING PROTEIN
ISOLATED FROM TIIE PLANT Blyonia Dioica Cross Reference This application is a ~ntinll~tirin-in-part of U.S. patent ~ A 11' " , Serial No. Og/141,891, filed October 25, 1993, the content thereofis hereby i-~cu~lJu--l~d by reference in its entirety.
0 ~ Field of the InventiQn The present invention relates to the isolation and ~ of a novel ibOaull~ c~iv_L;..g protein from the plant Bryonia dioica. The r l . ". " .... ,~ Jl ;.1r sequence encoding the protein and its amino acid sequence have been ~i~t~i~nin~ri The invention also relates to imm~lnr c.r njll~sit~c comprising the new protein and antibodies ; ...,.,...~lr~ specific for various tumor-associated antigens and to ll ' ' ''~uullailu1Ltd fiusion proteins having ribosome-i-.AcLivc.ii--~ activity and the ability to target specific cells. Methods for the Ir~ expression and chemical synthesis of this protein are considered part of the present invention. Use of these ;", ~ r~ g~ and 20 toxin fusion proteins in the treatment of cancer and as an active agent of various .r lr~g;~ C~ .r~ i5 al50 con5idered part of the present inventiOn.
Back~round of the Inventlon .

Proteins which inhibit protein synthesis have been isolated from various organisms including plants, bacteria and fungi. These protein toxins are thought to be produced by the organisms in order to provide a selective advantage for the growth of the organisms that produce them. Despite the divergent evolutionary b~.,l~l uull~ of the organisms in which these protein tûxins are fûund, mûst tûxins have strikingly sirnilar 1. . . h , ~ ~ of ~1 74943 action. One particular group of toxins exerts its action by blocking protein synthesis either by directly modifying eloneation factor 2 (EF-2) or by modifying the ribosome itself so that EF-2 caMot function in protein synthesis. This class of toxins, ribosome-inactivating proteins Q~s), can be isolated from plants of several families Plant ribosome-il.a~,~ivaLil,~ proteins have been divided into two groups based on their structure. Type I ribosome-inactivating proteins (type I RlPs) contain a single chain that has ribosome-i.la.,LivdLi.,~ activity. Examples of type I R]Ps include gelonin, saporin, r,~ and bryodin. Type 11 ribosome-illacLi~a.illg proteins (type II RlPs) are comprised of two chains, an A chain that is able to inactivate EF-2, and a B chain, that o contains a cell binding domain having lectin-like properties. The binding domain enables type 11 RlPs to bind many cell types and to kill those cells. Examples of type II RlPs are ricin and abrin.
Although the two types of ribosome-i..a.,Li~alil~g proteins differ in their structures, both types inhibit protein synthesis by ;--&~,~iv~L;-lg the 60S subunit of eukaryotic ribosomes through cleavage of the M gl~ ~os;~;c bond of the adenine residue at position 4324 of 28 S
rRNA (Endo and Tsurugi 1987, J. BioL Chem. 262:8128-8130; Stirpe, F. et al. 1988, NucL Acid ~es. 16:1349-1357).
Ribosome-inactivating proteins haYe been isolated from several families of plants includingtheCaliu~llJ" e, Cù~ul~ cedc~Eu~)llulb;a~ candPhy~ arr~rP~p The 20 toxins have been isolated particularly from the root, seeds and leaves of the plants.
Comparisons have been made of the N-terminal amino acid sequences of RlPs isolated from the seeds of Gelonium multif lorum ~ r~ r ~ r), A~; "lica charanlia (Cuuulb;~a~at), Bryonia dioica (Cuuulb;~a~,~a~ ), Saponaria officinalis (saporin-Sa, saporin-Sb, saporin-6a, saporin-6b) (Càliu~llylla~,~&~) and from the leaves of Saponaria 25 officinalis (saporin-l). Complete amino acid sequences have been determined for a Type I
RIP from Tri. h~u"J.~ Iarilowii maxim and from Barley seed protein synthesis inhibitor.
These cr." ,~ show that at least the N-terminal regions of the toxins bryodin and momordin (members of the Cul uu~b;~ceac family) show a high level of similarity with ricin A chain and with gelonin which are members of the EulJllolb;d~,~,ac family. The ~1 21 7~943 similarity is thought to be a rf~n~rlrnre of a similar evolutionary origin. Very little similarity was found between R~l?s of the Cucurbitaceae and Euphorbiaceae families and those ofthe Pl.y~ r~ or Cariophyllaceae families (Mont~cl~crhi et al.~ 1989, Inf. J.
Peplide Protein ~es. 33:263-267). Although similarities are found in the amino acid 5 sequences of the N-terminal regions of RlPs isolated from the same species, many differences do exist ~Gl Li~,ulGIly between toxins isolated from different tissue of the same plant.
A plant protein toxin designated bryodin was initially identified as a 27-30 kDal protein isolated from the root of ~ryonia dioica (United Kingdom Patent Application GB2194948, published March 23, 1988). The toxin is a type I l;boa~ G~livGii.. ~protein having a single chain and a mechanism of action which inactivates ribosomes by blocking productive .. .l rl .., .l lol-c with elongation factor-2. In not having a cell binding domain, bryodin, like the other type I RIPs, does not normally bind to -- ~ cells.
The protein has been shown to have a molecular weight by gel filtration of about 27,3 00 daltons and abotlt 28,800 daltons by polyG.,l~lG.l.;de gel ch.~llu~hc~ ;a~ and an isodectric point of 9.5. This toxin was found to inhibit protein synthesis in the rabbit ~ ,ulo~
Iysate system with wheat germ ribosomes at 3.6 ng/ml (IDso) and an LDso in mice of 14.5 mg/kg when adll... a~ d ;ll~l G~ O.~GIIY The N-terminal amino acid sequence has been determined to be Asp-Val-Ser-Phe-Arg-Leu-Ser-Gly-Ala-Thr-Thr-Thr-Ser~Tyr-Gly-Val-Phe-Ile-Lys-Asn-Leu-Arg-Glu-Ala-Leu-pro-Tyr-Glu-Arg-Lys-val-Tyr Asn-Ile-Pro-Leu-Leu-Leu-Arg-His-Xxx-Ile-Gly- (Seq. I.D. #8) A second ribosome-;l~G~,~ivGL;Ilg protein has been isolated from the leaves of B. dioica (European Patent Publication EPO 390 040, published October 3, 1990). This molecule has been described as having a molecular weight of 27,3 00 daltons by gel ... ..... . . . . . . .. .. ....

WO95111977 2l 74~43 PCTIUS94/12382 filtration and 28,800 daltons by pol.y~ .lid~ gel clc~L.ul,h~,l., ,i " and an isoelectric point of 9.5 and has been designated bryodin-L. This form of bryodin was found to inhibit protein synthesis in a rabbit reticulocyte Iysate system with an ECso of 0.1 nM (3.6 ng/ml) and has an LDso in mice of 10 mg/kg when a.l..li- i;,~c- cd ;IlLla~J~,. ikn~al ~y . An amino acid 5 analysis was also provided, but no amino acid sequence has been disclosed.
Ribosome-i.lc.,~ivdli.,g proteins are of interest because of their usefulnèss as(..,."1,.. ,1~ of ~ lr.x;..c " Immunotoxins are hybrid molecules consisting of atoxie moiety linked to an antibody eapable of selectively directing the toxin to a specific target eell. Potential target cells include harmful cells, ie., neoplastic, virally infected, 0 immllnnrnmretr~nt or parasitic cells T., ,, .,,1, ,. ~l l )Xi,.~ as deflned in the present invention can be chemical conjugates of a cell-specific ligand linked to a toxic molecule, such as a ribosome-i..a~,LivdLi..g protein The fact that many different libosu~.lc- ~ -g proteins are known and that new toxins are being discovered provides a variety of toxic moieties which have varying levels of intrinsic toxicity on whole cells when .. .~.. j ,g, 1 ~d and 5 provide an available source of alternative toxins should the patient develop an immune response during long term in vivo treatment to the originally au..li. isLc.~d ;...~ J~;
In addition, some 1111 Il -lL~-x;- ~, saporin 6 and an anti-Thy 1.1 antibody or its F(abl)2 fragment, were more toxic than free toxin providing a need for new and different toxin molecules.
The present invention provides a novel plant protein toxin isolated from Bryoniadioica we have designated bryodin 2, which is .~ Ir from bryodin and bryodin-L
by its n~ r,~ Lirl~ sequence, amino acid sequence, amino acid .~ , toxicity in arlimals and; ~ n~ ly Bryodin 2 provides a new ribosome-i..~..,Liv~.li..~ protein that ean be used to form additional and possibly better ;".,..., ... ,1 ..x;, .~ and toxin fusion 25 molecules for use in r. ." . .. ,~ ,l - 1 . Ar ~ O~ for use in treating caneer, eertain viral infeetions, modulating the immune response, and other diseases.

WO 9S111977 , _ PCT/US94112382 Summ~ry of the Invention The present invention comprises a novel ribosome~ iv~lLil.g protein comprising a single-chain protein having a molecular weight of about 27,000 daltons by poly~ Lulli.lc gel cl~,~,ll upllul ~,D;a under reducing and non-reducing conditions, an ECso of aboutO.017mMinarabbitreticulocytelysatesystem,anLDsoinmiceofgreaterthan 10 mg/lcg when a~ dV.,ll~JUDI~ and about 8 mg/kg when administered ciiLu~ lly. The ribosome ii~ ,Livo.L;llg protein ofthe invention further comprises an amino acid ~ o,~ determined on a residue per mole basis COIIIIJI ;D;II~;.
Lys 0.4 Ala 28.7 His Betow 1/2 Cys Below detection detection Arg 8.5 Val 34.2 Below Asx 14.0 Met detection Thr 13.1 Ile 23.3 Ser 6.5 Leu 28.3 Glx 38.2 Tyr S.O
Pro 15.0 Phe 18.5 Not Gly 11. I Trp determined This novel ribosome-inactivating protein was isolated from the plant B~yonia dioica, and has been designated bryodin 2. Bryodin 2 differs from ribosome-inactivating proteins 5 previously isolated from B. dioica and other plants in its nucleotide and amino acid sequence, and in its amino acid ~ , protein synthesis inhibitory activity and illllllu,lu~ l,L;v;Ly in various biological assays.
A second embodiment of the present invention comprises an isolated nliE~nn~l~lPoti~P sequence which encodes the l;lJùsoll~ v~i;llg protein isolated from WO 9S111977 , PCTIUS94/12382 ~yonia dioica having the amino acid sequence of bryodin ~ as depicted in Seq. ID. #15, or a comrlPmPnt of the isolated ol;~ u-,lev~ide. In particular the isolated r,l;~ f=
sequence can comprise the nli~,~"",rlr~,l;,~f sequence depicted in Seq. ID #14 or a fragment thereof which encodes a protein capable of ;lla~,~iv~ g a ribosome and 5 preventing protein synthesis.
In another ~IllI,o~;lll~ll~ of the present invention, the lil,osu~ va~ g proteincomprises an N-terminal amino acid sequence comprising the following contiguous amino acid sequence:
lo 1 5 10 Val Asp Ile Asn Phe Ser Leu lle Gly Ala Thr Gly Ala Thr Tyr Lys Thr Phe Ile Arg Asn Leu Arg Thr Thr Leu Thr Val Gly Thr Pro Arg (Seq. ID #1).
20 The li~osu. f~ i"~tivating protein can also further be comprised of a contiguous internal amino acid residue sequence of:

(a) Leu Pro Tyr Gly Gly Asn Tyr Asp Gly Leu Glu Thr Ala Ala Gly Arg (Seq. ID #2);

(b) Glu Asn Ile Glu Leu Gly Phe Ser Glu Ile _ W~95/11977 , 2 ~ 7 4 ~ 4` 3 PCTNS9~/12382 Ser Ser Ala Ile Gly Asn Met Phe Arg (Seq. ID #3);

(c) Phe Arg His Asn Pro Gly Thr Ser Val Pro Arg Ala Phe Ile Val Ile Ile Gln Thr Val Ser Glu Ala Ala Arg Phe Lys Tyr Ile Glu Gln Arg (S~q. ID #4);

(d) Tyr Ile Glu Gln Arg Val Ser Glu Asn Val Fly Thr Lys (Seq. ID #5);

(e) Phe Lys Pro Asp Pro Ala Phe Leu Ser Leu Gln Asn Ala Trp Gly Ser Leu Ser Glu Gln

2.~ 7 4~ 43 ~

Ile Gln Ile Ala Gln Thr Arg Gly Gly Glu Phe Ala Arg Pro Val Glu Leu Arg Thr (Seq. Il) #6); or (f) Leu Arg Thr Val Ser Asn Thr Pro Thr Phe Val Thr Asn Val Asn (Seq. ID #7).
In yet another ~ bo~ lwlL of the present invention, methods for the . ~ 5 expression of the ribosome~ Liv~Li..g protein of the present invention are described. The ly produced protein can be bryodin 2, fragments or derivatives of bryodin 2 having ribosome-;l.a~,Livd~i..g activity. The methods comprise preparing cl~ ly or genomic DNA which encodes bryodin 2, fragments or derivatives thereof, c~ a vector comprising the coding sequence operatively linked with ll A 11`' ' ;1ll ;~1' ' -I and 20 ~ elements necessary for expression in a host cell, ~ .. ,- the host cell with the expression vector, incubating the L,.,..~r~ host cell under conditions conducive to expression of the inserted coding sequence, and isolating the expressed ribosome V~.Lil-g protein.
In a further t...bo.li...~ , the ribosome-;..~ cli~ lg protein of the present invention 25 canbeusedtoforman; ",.",~ ; ortoxin-ligandconjugate. The; ~.",. .l/.~;
comprises a ligand or molecule that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a target cell population linked to the toxin. Ligands of the invention can be an ;" ." " gl~b l; ,, adhesion molecule, or a polypeptide, peptide or non-peptide ligand. Preferably, the ligand can be, but is not limited ... ... .. .. . . _ ... _ . . _ _ _ _ _ _ . . . .

wo 95/11977 2 i 7 ~ 9 4 3 PCT/US94112382 to, transferrin, an epidermal growth factor, bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, interleukin-2, interleukin-6, a Ll ,"ru~ "g growth factor, steroid, carbohydrate or a lectin. TmmllnnglA~lllin molecules specifically illllllU~ul~ .LivA
witn a tumor-associated antigen are particularly preferred. The ;, ", . " " ,..~1~.1 " .';, can be an 5 antigen recognizing fragment of an intact ;. ",, " " ,oglubu!~ ,, a chimeric antibody, or a hybrid antibody. Il~lllulloglobu!;.l~ specific for Lewis-Y related antigens which are intrrnAli7Pd by tumor cells are of particular interest in the present inYention. Specifically, a preferred c:lllboLI;,l.,,..~ of the present invention comprises the chimeric BR96 imm~lnf~ ' ' as produced by the hybridoma deposited with the American Type Culture Collection and 0 designated ATCC ~3Blû46û.
In another .,,,l,oll;ll~ ofthe present invention, the toxin and/or toxin-ligand conjugate of the present invention can be formulated to form a rl~A 1111-' .~ ~ 11~ .AI
C~ pl .A. I l ~ A"" ~ ~I Irl~ I ;fl~C of the present invention preferably comprise bryodin 2 or bryodin 2-ligand conjugates and a physiologically acceptable or ;.Alcarrier. Suchc,~..,l",~;l.,~ccanalsoincludevariousbuffers,excipients, additives and other molecules to stabilize the Il~ AI cu."~us;iiû.i.
In yet another rlllllO,I;,.,...l, the ribosome-;"AA~,Li~G~i"~ protein ofthe present invention can be used in methods for killing a target cell. Such a method comprises contacting the target cell with an effective amount of a toAxin-ligand conjugate comprising 20 the ribosome-ill_~,Li~Li--g protein and a ligand specific for the target cell. The toxin-ligand conjugate is contacted with the target cell for a time sufficient to kill the target cell. In a preferred ~",l~oJ;"I".lL, the toxin-ligand conjugates comprise bryodin 2 and thelullogl~bul;.. chimeric BR96, which, when contacted with tumor cells expressing the BR96 antigen, kills the tumor cells.
25 In still yet another .".lI,~d;ll.... ,llL, the ribosome-i~ .Li~AALi~g protein of the present invention is used in a method for inhibiting the l~ulir~ldLioll of ' tumor eells.
The method comprises the steps of eontaeting the mA nm~l;AA tumor eells with a l ." eomprising the ribosome-inactivating protein of the present invention conjugated with a ligand specific fûr a tumûr-assûciated antigen at a 1~ ul;r~,dl;ùll-WO 95111977 21 7 4 9 ~r 3 PCT/US94/12382 inhjbiting concentration for a time sufficient to inhibit the proliferation of the m~lmm~ n tumor cells. As above, in a most preferred w.,I,odi,..~,..L, the ..".I,n~ . comprises bryodin 2 and the immllnngln~ n chimeric BR96.
Brief Descrintion of the Drawin~s Figure 1 provides results of the absorbence reading from CM-Sepharose cluvlll~:L~ldlully of protein isolated from the root of Bryonia dioica Figure 2 is the result of SDS-PAGE analysis of fractions 19 through 27 from the o CM-Sepharose ~,lu u~ u~;l di,hy separation. Lane M contains molecular weight standûrds:
ovalbumin (43,000 mw), carbonic anhydrase (29,000 mw), ~ ,gln~ :, (18,000 mw), Iysozyme (14,000 mw), bovine trypsin inhibitor (6,000 mw), and insulin (2,000 mw).
Figure 3 is a chromatogram obtained firom a TSK-3000 size exclusion column.
Frûctions containing the 27 kDa band were pooled from the CM-Sepharose 15 ~luullll~Lu~ldLJll~l separation and cu~,L.dLt;d to less than 8 ml. The culll~ dLe was applied to the column and absorbence monitored at 280 nm.
Figure 4 illustrates the result obtained for SDS-PAGE analysis of fractions 58 through 64 from size exclusion c]llullldLu~ld~Jlly of the partially purified bryodin. Lane M
contains molecular weight standards: ovalbumin (43,000 mw), carbonic anhydrase ~o (29,000 mw), and ~ ~rtn~lnblllin (18,000 mw).
Figure 5 is a rnmrs~ri~nn of the similarity between the N-terminal amino acid sequence of bryodin 2 and other plant toxins. Bryodin 2 (BD2); bryodin 1 (BDl; Seq. I.D.
#8); ricin A chain (E~; Seq. I.D. #9); a--..oi.~u..,l.~.i,. (aMMC; Seq. I.D. #10);
L~ .n~,,ll.;.. (TCS; Seq. I.D. #11) and luffin A (Seq. I.D. #12).
Figure 6 provides the amino acid sequences obtained for various fragments of the27,000 protein band isolated from the roots of BrJlonia dioica, after treatment with cyanogen bromide and certain proteases.
Figure 7 illustrates the alignment of amino acid sequences obtained from peptidefragments of bryodin 2 with the plant toxin momordin.

WO 95/11977 2 1 7 ~ 9 ~ 3 PCT/US94/12382 ~ igure g illustrates ELIS~ binding of anti-BD2 antibody (50-44-3) to i" "~ f ~1 ribosome-inactivating proteins. Detection was done with goat anti-mouse IgGI HRP.
BD2 ([1), BDI (--), ricin A chain (--).
Figures 9A through C illustrate the purification of chiBR96-;"... ,. l,.. ,l. ,~;
5 conjugates. BR96 and BD2 were chemically conjugated via a hindered disulfide linkage and purified by a two-step chromatography process. Figure gA is the ~,1".,~ ,h.~profile from the gel filtration column of chiBR96-BD2 conjugate. Fractions 45-55 are the conjugate and unreacted antibody; fractions 64-74 are unreacted antibody. Figure 9B is theNaCI elution profile of chiBR96-BD2 fromBlue-Sepharose (0.4 MNaCI, fraction l;
o 0.8 M NaCI, fractions 2-8). Figure 9C is the Coomassie Blue stained SDS-PAGE analysis of fractions of the Blue-Sepharose eluted material (4-12% non-reducing polyG~.lyl~...;d~
gel). Lanes 1-4 correspond to fractions 1-4 from panel B, Lane 5~ 1 chiBR96.
Figure 10 illustrates the binding activity of BR96-BD2 and BR96-BDI
i.. u.. vlv~i.. conjugates. Binding of BR96-;. "" " " " .~ was determined using H3396 5 cell ~ Specific antigen binding was detected with goat anti-human IgG
h~r.~r~ h peroxidase. Data represents duplicate data points. Chimeric BR96 (chiBR96, ), chiBR96-BD2 (O), chiBR96-BD1 (~), BD2 (~), BDI (--).
Figures 1 lA and 1 lB illustrate the ;ylulv~i~.;ly of chiBR96-BD2 and chiBR96-BD1 " ". "., ,l ,l r~ conjugates. Cell killing was determined following incubation of 20 chiBR96-BD2 and chiBR96-BDI ;".. """~ .l.. ~;., conjugates with (A) H3396 breast carcinoma cells (antigen positive) and (B) H371g colon carcinoma cells (antigen negative) for 96 hours. Cell killing was determined by measuring calcein-AM hydrolysis into fluorescent calcein. ChiBRg6, (--), chiBR96-BD2 (O), chiBR96-BD1 ( ), BD2 (C), BD1 ( ).
Figure 12 provides the ollg,~""~l. vl;flP sequence encoding bryodin 2 (Seq. ID# 14) and the putative amino acid sequence encoded by the ~il r.. - ~ l. vl; 1r sequence (Seq.
ID#15. The o~i~nnllrleotirl~ sequence provides for the translation of a mature protein of about 261 amino acid residues with a 21 amino acid residue signal sequence.
Il WO g511 L977 ~ 7 4 q 4 3 . PCTIUS94/12382 Figure 13 illustrates an alignment ofthe amino acid sequence obtained for bryodin 2 with the plant toxin momordin.
DetAiled Description of the SDecific Embodiments The present invention relates to a novel ribosome-il.a.,~iv~ g protein toxin isolated from Bryonia dioica, we have designated bryodin 2, to methods of producing bryodin 2 by ~D.l~.".Li~llal l inchf-miAAI or It,~,ullllJillall~ means, to C~J ,l)o~ll;nAc comprising the toxin, and to therapeutic methods utilizing the toxin as an immune conjugate or a toxin lo fusion molecule.
Bryodin 2 (BD2), a novel ribosome-inactivating protein, is isolated from the roots o f Bryonia dioica. BD2 exhibits toxicity to cells similar to other plant ribosome-i~G~,~iv_~ proteins, suggesting that it may be useful in the killing of cells, ~A ~ Li~ulA-l ly if directed to a deflned cell population by the ligand of a cell-specific molecule. Such ligands can include an antibody, a ligand of a cell-surface receptor (i.e., transferrin, heregulin, and others well known to the skilled artisan). BD2 can also be used in the f.~ . ." of conjugates or fusion molecules comprising the ligand of a cell-specific molecule and the toxin which would be useful in the treatment of a disease state.
Purified bryodin 2 has been detected as a single band of ~ .Ai..._Lel.~ 27,000 20 dalton molecular weight under both reducing and non-reducing conditions. BD2, therefore, comprises a single chain polypeptide.
A partial primary structure of BD2 described herein has been determined by aminoacid sequencing of various peptide fragments generated by specific chemical and enzymatic cleavage of BD2. Sequence analysis revealed that BD2 is a type I ribosome-i.~ iYdLi.lg 25 protein having some similarity with, but distinct from, other ribosome-i.._~.~ivd~;.lg proteins ofthe Cu~u~ e~c family including bryodin, l~;~ l,n~A"ll,... and a-,-,o...u-e.._.i..
(1`~1~ ,"If~ ; et al., 1989, In~. J. Peptide Protein Res. 33:263-267), All of these proteins display certain comrnon properties ~LAI _-,Lel iaL;~; of type I l ;I,o:,oll,~, illA~.~iVA ~ proteins, such as being comprised of a single-peptide chain, a molecular weight of between 25 and WO 95111977 . 2 ~ ~ ~ 7 ~ 3 PCT/US94~12382 .
30 kDa and having an isoelectric point of dlJplu~ lJ 9.0-lû.0 (Stirpe and Barbieri, 1986, ~EBSletl. 196:1-8; rlmenez and Vasquez, D., 1985, ~nn. Rev. Microl~ioL 39:649-672).
The amino acid sequences have been confirmed by the cloning of the gene 5 encoding bryodin 2 from the leaves of Br)~onia dioica A complete C~l;b' l 1. I P- .1;~1( sequence encoding mature BD2 and the putative signal sequence is provided in Figure 13.
Bryodin 2 inhibits protein synthesis (ECso=0.017 nM) in a cell-free in vifro translation assay using rabbit reticulocyte Iysate. Also, BD2 is toxic to mice with LD50 values of greater than 10 mg/kg when administered ;llL~ lluualy and about 8 mg/kg when 10 a.l.. i.~ d ;~ ;lr.. ~ ^lly, Toxicity is most likely due to liver damage as seen hiCt~chPmir~lly by the presence of liver lesions and by an increased liver protein in a blood chemistry screen (data not shown). In çr mrArienn the LDso Of bryodin I has beenreported to be 14.5 mg/kg, i.p. (Stirpe et al., 1986, Biochem. J. 240:659-665).
The production and use of derivatives, analûgues, and peptides related to bryodin 2 5 are also envisioned and are within the scope of the present invention. Such derivatives, analogues, and peptides which exhibit lil.o~u--lc- - - I;vaiillg ability to inhibit protein synthesis can find uses and Arrlic ~tirlnc in the treatment ûf a wide variety of diseases.
Such derivatives, analogues, or peptides can have enhanced or diminished biological activities in cl .. ~ IIIAI ;~UII to native BD2.
BD2-related derivatives, analogues, and peptides of the invention can be produced by a variety of means known in the art. Procedures and ~ ;. . c of both the genetic and protein levels are within the scope of the present invention.
Bryodin 2 is produced by cells of the rûot, leaves, and berries of Br~onia dioica and can be purified to hull~og~ y firom extracts of plant tissue. Methods used to purify bryodin 2 are those commonly used in biuell~ llfially and can include various f u of c~ gr;;~ U~ ,y, andpolyacrylamidegelcl~L, ulJIIull,a;a. The ~111 Ull~:LiU~ y methûds used can include, but are not lirnited to, c.. ., l, . I ;r ~ of ion exchange, gel perm~tir~n. and affinity "1., Ulllr Lugl ~1,~. Affinity i~L~ .Liu~s including ol;i~ y~ yorûtheraffmity;ll~riAll;llll~arecûnsideredaspar~tofthe WO 95/11977 ~ ~ 7 4 q 4 3 . PCTIUS94112382 prescnt invention. All of the chromatography methods can include both low pressure and high pressure methodologies.
Alternatively, BD2 can be produced by l~1ulllb;llallL DNA techniques or chemicalsynthetic methods. To produce BD2 by I ~culllb;l~all~ methods, messenger RNA (mRNA) 5 for the preparation of ~ A I y DNA (cDNA) can be obtained from cell sources that produce BD2, whereas genomic sequences for BD2 can be obtained from any cells ofBryonia diofca regardless of tissue type. For example, roots of B. dioica can be utilized either as the source of the coding sequences for BD2 and/or to prepare cDNA or genomic libraries. Genetically-engineered microorganisms or cell lines ~,~"i,r.""..,d or transfected lo with total DNA or RNA from a source line can be used as a convenient source of DNA for screening.
Either cDNA or genomic libraries can be prepared from DNA fragments generated using techniques well known in the art. The fragments which encode BD2 can be identified by screening the prepâred libraries with a nucleotide probe which would encode 15 an amino acid sequence 11- " "- lr~ to a portion of the BD2 amino acid sequence in Figure 5 (Sequence ID#s 1-8). Although portions of the coding sequence may be utilized for cloning and expression, full length clones, i.e., those containing the entire coding region for BD2, may be preferable for expression. To these ends, techniques well known to those skiUed in the art for the isolation of DNA, generation of appropriate fragments, by 20 various methods, c~ u.,~iol~ of clones and libraries, and screening ~ can be used. See, for example, the techniques described in Sambrook et al., 19~9, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, NY.
Due to the degeneracy of the nucleotide coding sequences, alternative DNA
sequences which encode analogous amino acid sequences for â BD2 gene can be used in 25 the practice of the present invention for the cloning and expression of BD2. Such alterations include deletions, additions or ~ of different nucleotide residues resulting in â sequence that encodes the same or a functionally equivalent gene product.
(See Example 9, and Table 3 for specific probes.) The gene product may contain deletions, additions or ~1 .l ,~:; l l l ;. ~"~ of amino acid residues within the sequence, which . ,,, , ., , ,,,, . , .. ,, .. , .. , , .. ., .. . . , .. , .. . , . . , _ .. _ . _, ..... ... .. .......... .....

wo 95~11977 . 2 1 7 ~ ~ ~ 3 PCTIUS94112382 result in a silent change thus producing a bioactive product. Bioactivity in this context is measured by the ability of the gene product to inhibit protein synthesis.
~ ny amino acid ~l lhstit~ltinn~ can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity/hydrophilicity and/or the A l I l~ nature of the s residue involved. For example, negatively charged amino acids include aspartic and glutamic acid; positively charged amino acids include Iysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following:
leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine;
p~ L.la~ , tyrosine.
In order to express a biologicdlly active bryodin 2, the nucleotide sequence encoding BD2, or a rull~.Liul~ally equivalent nucleotide sequence, is inserted into an d~lJlu~u~iaie vector, i.e., a vector which contains the necessary elements for the llall>.,li~Liull and translation ofthe inserted coding sequence. Modified versions ofthe BD2 sequence can be engineered to enhance stability, production, l."l;r..Al;~"-, yield or 5 toxicity of the expressed product. For example, the expression of a fusion protein or a cleavable fusion protein comprising BD2 and a ll.,~eluloguu~ protein can be engineered.
Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity ~ U ., l ~ 'ly, e.g, by ;111111~ A1;~ 1~ on a column specific for the heterologous protein. Where a cleavage site is engineered between the BD2 moiety and the 20 I..,iG ulo~;uu~ protein, the BD2 protein can be released from the cl,.. ,,..~ , column by treatment with an appropriate enzyme or agent that disrupts the cleavage site (e.g, see Booth et al., 1988, ImmunoL LelL 19:65-70; and Gardella et al., 1990, J. BioL
Chem. 265:15854-15859).
Methods which are well known to those skilled in the art can be used to construct 25 expression vectors containing a BD2 coding sequence and a~ )1 id~C
tr~Anc~ rirtif.nAl/ll ",l ,laiio,,a~l control signals. These methods include in vi~ro ~ r J
DNA techniques, synthetic techniques and in vivo IC~ UI~ l..Li., techniques. See, for example, the techniques described in Sambrook et al., 1989, Molecular C70ning ,4 LaboratoryMan1lal, 2nd~d., Cold Spring Harbor Laboratoly, NY.

W09S/11977 ~'l 7 $9 43 PCT/US94/12382 A variety of host-expression systems can be utilized to express the BD2 coding sequence. These include, but are not limited to, microorganisms, such as bacteria .1 wjth a lr~1 111111111~11l bacteriophage DNA, plasmid DNA or cosmid DNA
expression vector containing the BD2 coding sequence; yeast l,..,~."".~;1 with 5 ~c~,u~ L yeast expression vectors containing the BD2 cûding sequence; plant cell systems infected with, r~ virus expressiûn vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or Ll d~ ,d with, C~Ulllb;~ plasmid expression vectors, such as Ti piasmid, containing the BD2 coding sequence. To use mqmmql;qn expression systems, the BD2 ribosome-inactivating activity would have to be blocked or lO masked until Iysis of the host cell or secretion of BD2 into the culture medium to protect the host cell from the toxin effects of BD2 or a mutant host cell resistant to the bryodin must be used.
Depending on the host/vector system utilized, any of a number of suitable ,,, and translation elements including constitutive and inducible promoters, 5 ~ ", enhancer elements, transcription terminators, etc., can be used in the expressionvector(see,e.g,Bitteretal., 1987,MethodsinEnzymoL 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of .lr~ ,~,pl.,~ ; plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
Promote}s produced by Ic~.ulllb;llall~ DNA or synthetic techniques can also be used to 20 provide for controlled and high level ~ ~."~ iûl~ of the inserted BD2 coding sequence.
In bacterial systems, a number of expression vectors can be aJ~ ~."~Ju~l~
selected depending upon the use intended for the BD2 expressed. For example, when large quantities of BD2 are desired, vectors which direct the expression of high levels of protein product, possibly as a fiusion with a lly ilu~.hùb;c signal sequence, which directs the 25 expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified may be desired. Certain fiusion protein engineered with a specific cleavage site to aid in recovery of the BD2 may also be desirable. Such vectors adaptable to such mqnirlllqtion include, but are not limited to, the pET series of E coli expression vectors (Studier et al., l990, Metho~s in EnzymoL 185:60-89).

.. .. , . ... ., .. ... , .. . . . .. , .. ... _,, .. . ., .. , .. . = , . , , ,,, _, .. .

WO95111977 ~ 4~3 PCT/US94112382 In yeast, a number of vectors containing Co~ iLuLive or inducible promoters can be used. For a review, see CurrentProtocols inMolecularBiology, Vol. 2, 1988, ed.
Ausubel et al., Greene Publish. Assoc. & Wiley T~lr-~ ~,,;. .Ir ~, ch. 13; Grant et al., 1987, "Expression and Secretion Vectors for Yeast," in Methods in Enz,vmol. 153:516-544;
Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, "Heterologous Gene Expression in Yeast," in Methods in EnzymoL 152 :673-684. A
CO~ u~iYe yeast promoter such as ADH or Leu2 or an inducible promoter such as GAL
can be used ("Cloning in Yeast," ch. 3, R. Rothstein In: DN~ Cloning, Vol. 11, APractical Approach, Ed. D.M. Glover, 1986, IRL Press, Wash. D.C.). Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast ,lu v~lo~Ull~
In cases where plant expression vectors are used, the expression of the BD2 coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and l9S RNA promoters of CaMV (Brisson et al.7 1984, Na~ure 310:511-514), or thè coat protein promoter to TMV (Takamatsu et al., 1987, EMBO J: 6:307-311) can be used. Alternatively, plant promoters such as the small subunit of RUBISCO(Coru~zi et..al., 1984, EMRO J~ 3:1671-1680; Brogli et al., 1984, Science 224:838-843);
or heat shock promoters, e.g, soybean hspl7.5-E or hspl7.3-B (Gurley et al., 1986,MoL
CelL BioL 6:559-565) can be used. These constructs can be introduced into plant cells 20 using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA l", r~" ", .,;,., IIU~,IU;II;C~,L;VII, CI~LIVPVI~IL;O~ and other techniques well known to the skilled artisan.
See, for example, Weissbach & Weissbach, 1988, Me~hodsforPlantMolecularBiolog,v,Acadernic Press, NY, Section VIII, pp 421-463; and Guerson & Corey, 1988, Plant MolecularBiology, 2d ed., Blackie, London, Ch. 7-9.
2s Other expression systems such as insects and mq nn ~ host cell systems are well known in the art, but would have to be modified or adapted to produce a toxic molecule.
One potential approach to ,"r~ would be to isolate mutant insect or mqmmql;~n cell lines resistant to BD2, as mentioned above.

WO 95111977 '2 1 7 4 9 ~,L 3 . PCTIUS94112382 In addition to producing bryodin 2 by l~ .~.,..l IillAIII DNA techniques, BD2 can also be produced in whole or in part by solid phase chemical synthetic techniques based on the determined amino acid sequence (see, Creighton, lg83, Prote;n Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 50-60; Stewart and Young, l9g4, Peptide Synthesis, 2d Ed., Pierce Chemical Co.). This approach may be particularly useful in generating segments or fragments of BD2 corresponding to one or more of its biologically active regions.
Also within the scope of the present invention is the production of polyclonal and antibodies which recognize bryodin 2 or related proteins.
o Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of BD2. For the production of antibodies, various host animals ean be immunized by injection with the BD2 protein, or as BD2 peptide,. including but not limited to, rabbits, hamster, mice, rats, etc. Various adjuvants ean be used to inerease the imml nnlnpirAl response, depending on the host speeies, ineluding but not limited to, Freund's (eomplete and incomplete); mineral gels, sueh as aluminum hydroxide; surfaee aetive substanees, such as Iysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet l~ lo.,y~ l, d;~I;LI~ IVI~ and others well know to the skilled artisan.
A ~ n~ antibody imml~nnlr~ ly specific for an epitope of BD2 ean be prepared by using any of a number of teehniques known to the skilled artisan whieh provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique originally deseribed by Kohler and Milstein (1975, Nature, 2~6:495-497), and more reeent, ~ d; ~A" A 1l l '` of those teehniques.
Antibody fragments which eontain the idiotype of the moleeule ean be generated by ~5 known teehniques. For example, such firagments include, but are not limited to: the F(ab')2 fragments generated by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing disulfide bridges of the F(ab')2 fragments.

WO 95111977 , 2 ~ 7 ~ 9 ~ 3 PCT/US9~/1238~
.
In another aspect of the present invention, the bryodin 2, or a functional equivalent, can be used with a ligand for a cell surface receptor to target the toxin to a specific cell population as a toxin-ligand conjugate.
The skilled artisan . ~ the term "ligand" includes within its scope any s molecule that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a given target cell population. Tbis cell-reactive molecule, or ligand, to which the toxin is linked via a linker in the conjugate, can be any molecule that binds to, complexes with or reacts with the cell population sought to be thl~rqrc~ y or other vise biologically affected. The cell-reactive molecule acts to o deliver the toxin to the particular target cell population with which the ligand reacts. Such molecules include, but are not limited to, large molecular weight proteins (generally greater than 10,000 daltons) such as, for example, antibodies or adhesion molecules, smaller molecular weight proteins (generally, less than 10,000 daltDns), poly~ ,Lidc~, or peptide ligands, and non-peptidyl ligands.
The non-i,.. l,u.. o. uG~,Live protein, p~ idc, or peptide ligands which can be of use to form tbe conjugates of the present invention may include, but are not limited to, transferrin, epidermal growth factors, bombesin, gastrin, gastrin-reieasing peptide, platelet-derived growth factor, IL-2, IL-6, or tumor growth factors, such as TGF-c~ and TGF-~.
Non-peptidyl ligands may include, for example, steroids, ca.L,ol.y.l.dL,,~ and lectins.
The ;~ u~o~c~.LiY~ ligands comprise an antigen--c.,u~;.. i~;.. g ;".,.,., ~glnb.,l", (or antibody), or antigen-.~,o~..;,i.lg fragment thereof. Particularly preferred immunoglobulins are those i, " " " " ..~pl ~n~ _' which can recognize a tumor-associated antigen capable of int~qli7qfinn As used, "i " " "" "n~lol,_' " may refer to any recognized class or subclass of imm~lno~lobulin such as IgG, IgA, IgM, IgD or IgE. Preferred are 25 those ;" ." " " .~ gl. ,L."I . ~ which are within the IgG class of ;, . ", ,. .. "~glnL."lin~ The illLllUIlOglo' _' can be derived from any species. Preferably, however, the ul~globul;ll is one of human or murine origin. Further, the ;.,.,....,.ngl"LIul l may be polyclonal or ....~ 1, preferably mnnnrlnn W0951ll977 21 7 ~ ~ 4 3 PCT/US9~/12382 As noted, one skilled in the art will appreciate that the invention also ~
the use of antigen recognizing immunoglobulin fragments. Such; " " ", .~ glnb~ "fragments include, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen ~I;.,O~ll;~llg ;~ ulloglDbu!;.l fragments. Such ;" " ll' ...nglnL" ll;. l fragments can be prepared, for example, by proteolytic enzyme digestion, for example, by pepsin or papain digestion, reductive alkylation, or l~u~l~L~ L techniques. The materials and methods for preparing imm~lnnglnblllin fragments are well known to those skilled in the art. Seegenerally, Parham, 1983, J. Immunol. 131:2895; Lamoye et ai., 1983, J. ~mmunol. Me~hods 56:235;
Parham, 1982, J. ImmunoL Mefhods 53:133 and Matthew et al., 1982, J. ImmunoL
0 Methods 50:239.
The ;l l l l l ll l l ,nglnl .~,l; " can also be "chimeric" as that term is recognized in the art.
Aiso, the imml~nn~lnb~lin can be a "~ or "hybrid" antibody, that is, an antibodywhich may have one "arm" having a specificity for one antigenic site, such as a tumor-associated antigen, while the other arm recognizes a different target, for example, a second 1~ ceil type-specific receptor molecule. In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state.
Rifi.n~tinn~l antibodies are described, for example, in European Patent Publication EPA 0 105 360. Such hybrid or bifilnrtinn~l antibodies may be derived, as noted, either i,;olog;~ by cell fusion techniques, or chemicaily, especially with cross-linking agents or disulfide bridge-forming reagents, and may be comprised of whole antibodies and/or fragments thereof Methods for obtaining such hybrid antibodies are disclosed, for example, in PCT appiication W083/03699, published October 27, 1983, and EuropeanPatentPublication,EPA0217577,publishedApril8,1987,bothofwhichare .u,~Led herein by reference.
In addition, the ;IlllllU,.Og:~ ' may be a single chain antibody ("SCA"). These can consist of single chain FY fragments ("scFv") in which the variable light ("VL") and variable heavy ("VH") domains are linked by a peptide bridge or by disulflde bonds. Aiso, _, . .... ... . . . ... . .. . ... . . . . ... ..... . , .. .. = . . .

WO 95111977 2 ~ 7 4 9 4 3 PC,/US94/12382 the imml-nnFlnblllin may consist o~single VH domains (d~bs) which possess antigen-binding activity. See, e.g, Winter and Milstein, 1991, Nature 349:295; ~'llnrk~h~l)or et ai., 1990, f~jf.~hf~mi~fr~v 29:1362 and Ward et al., 1989, Nature 341:544.
A preferred embodiment of an immunologicai ligand as part of a ligandltoAvin s eonjugate for use in the present invention is a chimeric ~ n~ antibody, preferably those chimeric antibodies which have a specificity toward a tumor-associated antigen. As used herein, the terrn "chimeric antibodyl' refers to a, l l. ~f~nf ln~ ~l antibody comprising a variable region, Le., binding region, from one source or species and at least a portion of a eonstant region derived from a different source or species, usually prepared by 0 1--~ f~ DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred in certain ~, l ' of the present invention, particulariy human therapy. Such IllUli~ ilUlllo,ll chimeric antibodies are the product of eAvpressed ;"." ,. ,. ,nglf l,, ,l;" genes comprising DNA segments eneoding murine iJ~UllUll;)glOiJUIill variable regions and DNA segments encoding human; " ", .l " .nglob~
eonstant regions. Other forms of "chimeric antibodies" _.. ~ by the present invention are those in which the elass or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "elass-switehed antibodies." Methods for producing chimeric antibodies involve Co..~..llioil~l IC~,fJllliJ;llf.
DNA and gene ~ r~ I ;nl~ techniques now well known in the art. See, e.g, Morrison et al., 1984, Proc.Natl.. 4cad ScL USA81:6851;U.S.PatentNo 5,202,238,andU.S.
Patent No. 5,204,244.
d by the term "chimeric antibody" is the concept of antibody," that is, those antibodies in which the framework or 1~ A. ;Iy ,i. ;rl .""""~ regions" (CDR) have been modified to comprise the CDR of an 2~i imm~lnn~lnblliin of different specificity as compared to that of the parent ~ lgln~
In a preferred clllI,odilll~,l.i, a murine CDR is grafted into the framework region of a human antibody to prepare the "I~u~ .,l antibody." See, e.g, Riechmann et al., 1988, Nature 332:323; and Neuberer et al., 1985, Nature 314:268. Particularly preferred CDRs WO 95111977 ~ 1 7 '~ ~ 4 3 . PCI/IJS94112382 correspond to those ~ , C~ ;llg sequences recognizing the antigens noted above for chimeric and hifilnrti-~nAI antibodies.
One skilled in the art will recognize that a hifi~nr tion~l chimeric antibody can be prepared which would have the benefits of lower ;~ y of the chimeric or 5 humanized antibody, as well as the flexibility, especially for therapeutic uses, of the bifiln~ti~n~l antibody described above. Such l~;rl.~ chimeric antibodies can be synthesized, for instance, by chemical synthesis using cross-linking agents and/or .ulllbi~ llL methods of the type described above. In any event, the present invention should not be construed as limited in scope by any particular method pf production of an 0 antibody whether ~ chimeric, b; ~ " . I ;. .. ~l-chimeric, humanized or an antigen-recognizing fragment or derivative thereo ~ urther, as noted above, the ;,...."",~.gl-.l,"': ., or fragment thereof, used in the present invention may be polyclonal or ",.~ in nature. Monnrlnn~l antibodies arethe preferred ;llllllulloglo~ulil~, however. The preparation of such polyclonal or ml-nr~ nAI antibodies now is well know to those skilled in the art who are fully capable of producing useful immllnl glml ' which can be used in the present invention. See, e.g, Kohler and Milstein, 1975, Na~,~re 256:495. In addition, hybridomas and/or " " ,. .."l", ,.l antibodies which are produced by such hybridomas and which are useful in the practice of the present invention are publicly available from such sources as the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, or colll",~ ,l'y, for example, fromBoehringer-MannheimT~ rh~ mir~ P.O. Box 50816, T I I . l; A . . A ¦ I ~1 i ~, I N 4 6 2 5 0 .
A p~ iuul~lly preferred m~nr~lt)nAl antibody of the present invention is one that binds a tumor-associated cell surface antigen and is capable of;..l~ l,,Al;..l\ In a 25 particular embodiment of the present invention, the toxin is conjugated to the chimeric antibody BR96 ("chiBR96"), disclosed in U.S. Serial No. 07/544,246, filed June 26, 1990, and which is equivalent to PCT Published Application, WO91/00295, published January 10, 1991. ChiBR96 is an inti~rn~li7in~ murine/human chimeric antibody and is reactive with a fucosylated Lewis Y antigen expressed by human carcinoma cells, such as 2~

WO 9S/11977 2 1 7 4 ~ 4 3 PCr/US94112382 those derived from the breast, lung, colon, and ovarian carcinomas. The hybridoma expressing chimeric BR96 and identified as the chiBR96 was deposited on May 23, 1990 under the terms of the Budapest Treaty, with the American Type Culture Collection, and designated ATCC ~310460.
One of the preferred methods of making an ;~ u~ , of the present invention is by chemically u~; ~g~ e the bryodin 2 toxin with the ligand, preferably a .,,~
antibody or a fragment thereof, as described above. Many methods of chemical ~n~ grl;U,. are well known to the skilled artisan. See, e.g, Vitetta et al., 1987, Science 238:1098; Pastan et al., 1985, Cell 47:641; and Thorpe et al., 1987, Cancer o Res 47:5924). These methods generally conjugate the toxin and the antibody by means of cross-linking agents that introduce a disulfide bond between the two proteins.
T~ which have been prepared with ,u,.. eJu~,;blc linkages have been shown to be cullsis~ Lly less cytotoxic than similar toxins cross-linked by disulfide bonds.
One preferred method uses N-succinimidyl-3-(2~ .;.1yLl;Ll.;o)-propionate (SPDP) 15 and 2-; ", l. .~ hydrochloride (2IT). Other preferred reagents are sodium S-4-~ùcc;~ lu~ywlbu~yl-a-methyl ben_yl thiosulfate (SMBT) and 21T or ~u1c;.llll~uyluAy carbonyl-a-methyl-(2-~, y. ;d yll;~i.k,)-toluene and 2IT. Each group of reagents introduces a disulfide bond between the toxin and the antibody which is reducible, but the bond is also resistant to breakdown providing stability of the conjugate in vitro and 20 in ViYo. Upon intPm~li7~tioll into Iysosomes or endosomes by the target cell, the bond is reduced and the toxin enters the cytoplasm, binds elongation factor 2, disrupting protein synthesis.
Anûther preferred embodiment of the present invention is l e~
IIUIIU~U~ , partic~larly single-chain ;~ lc These molecules have the 2~ advantage over toxin-antibody conjugates (~ llul-u~ dl- ,) in that they are more readily produced than the conjugates, and l~olll~g~,llcou~ pC~ of toxin molecules are generated, i.e.l single peptide composed ofthe same amino acid residues.
The techniques for cloning and expressing DNA sequences encoding the amino acid sequences ~ Jull~l;llg tû a single chain derivative ûf a parental antibûdy are we]l ~3 wo95/11977 ~ 7 4q 43 PCI/US94/12382 known to the skilled artisan, as discussed above. Methods for dPtPrminin~ the nucleotide sequence and complete amino acid sequence of bryodin 2 are also described above.Various methods of ~,u"~LI Ul.,~;llg I ~C~ toxin fusion proteins are described in Pastan and Fitzgerald, 1991, Science 254, 1173; Siegall et al., 1988, Proc. NatL Acad. Sci USA
s 85:9738; Batra et al., l991, MoL Cell BioL 11:22ûû; O'Hare et al., l99û, FEBSLett.
273:200; Westby et al., 1992, Bioconj. Chem. 3:375.
The plant ribosome-inactivating toxin, bryodin 2, of the present invention is useful for therapeutic ArpliA.Ation~A, both in vitro and in vivo in its isolated form and as ligand-toxin conjugates and Ir~ toxin fusion proteins. Ribosome-i"A.,~iv.lL;"g proteinsisolated from C-l~,u~bil~-~,ed~ plants have found use as, among others, ab~lLiL.,;~t~, imml~nAIm~ At~rs, anti-tumor and anti-viral agents (Ng et al., 1992, Gen.
Pharmac. 23:575-59û) or as an anti-malerial agent (~morim et al., 1991, Mem. Inst.
Oswaldo Cruz 86:177).
Bryodin 2 is particularly usefiul as a ligand-toxin conjugate or a I c ~.. ", ,1. . - ,I toxin fusion protein since BD2 is less toxin than many other protein toxins and ribosome-i IA~.~iv~,lillg proteins that have been used to construct ;"""""~ x"~ and is p~ uLuly potent at inhibiting protein synthesis once inside the cell. Ligand-toxin conjugate and I C~,~JIIIII;II~III~ toxin fusion proteins can be used for either in vivo treatment of cells removed from the body or a patient to remove or kill a desired cell population prior to reinfusion of the remaining cells back into the patient or directly All,.,;.,: Irl ,"~ the l~ -toxin fusion into the patient.
For ex vivo uses, cells, such as bone marrow, may be removed from a patient suffering from cancer and the marrow purged by treatment with the ligand-toxin conjugate or fusion protein. Following treatment, the marrow is infused back into the patient (see, e.g, Ramsay et al., 1988, J. Clin ImmunoL 8:81-88).
For in vivo uses, the present invention provides a method for selectively killing cells, i.e., tumor cells, expressing the antigen that specifically binds the ligand, or functional equivalent of the ligand-toxin conjugate or fusion molecule. This method comprises reacting the toxin conjugate or fusion molecule with the tumor cell by wo 95/119~7 . 2 1 7 ~ 9 4 3 PCT/US94/12382 rl ;l lg to a subject a ph~ dccu~icdlly effective amount of a . .. " "l~t.~ . containing at least one ligand-toxin conjugate or fusion molecule of the present invention.In accordance with the present inYention' the subject may be human, equine, porcine, bovine, murine, canine, feline, and aYian. Other warm blooded animals are also 5 included within the scope of the present inYention.
The claimed invention also provides a method of inhibiting the ~ .lirc..llio.i of tumor cells, pd~ LicUI_- Iy mAmmAliAn tumor cells. This method comprises contacting the tumor cells with a p- ulirt~ ~.Lioli inhibiting amount (i.e., effective amount) of a tumor targeted toxin joined to a ligand specific for a tumor-associated antigen so as to 10 inhibit pl ~lirel ~LLiu~l of the mammalian tumor cells.
In one example, bryodin 2 is conjugated to the chimeric ,.., .... ,~ antibody BR96 (chiBR96) specific for the Lewis Y tiPt~-rmin ~nt and capable of ' ~ withinthe tumor cells to which it binds. Tumor cells were contacted with the chiBR96-BD2 conjugates in vi~ro at various dosages to determine an amount of chiBR96-BD2 conjugate 15 effective for cell killing. Eifrt.,li~ was deterimined in vitro by several methods 'cnown to one skilled in the art including ~;yLoLu~ ,;Ly assays.
The subject invention further provides methods for inhibiting the growth of human tumor cells, treating a tumor in a subject, and treating a ivl uli'c. aLiYt type disease in a subject. These methods comprise AA~ r ;1 l~ to the subject an effective amount of the 20 t.t)nAroeitinn ofthe invention. FX~ t)~-I;V~I from mammalian model systems for diseases such as cancer can be difficult in some cases. But, animals do provide more than just a iulI' yscreenofpotentialtherapeutic C~ J~.I;t~ Eachc~ ;,n~ whichis determined to have an effective dose in an animal model to inhibit the i!~lulirtld~;~lll of or 'cill a target cell in vivo d~ o~ c~ that the romrnQitinn is an active agent for inhibition 25 or killing. One of skill in the art can and does use this information to provide a basis for testing a ,- , ..~ ;;;-,, for c~.,li~ , in humans. Ail ~ ..., .I.o~;l ;n. ,C previously tested in animals demonstrated the requisite activity in humans. The only remaining question to be determined is any potential adverse effects from the co..li o~;Lh,l, particular to the human system and whether the co~ ,u~iLi~ is ultimately effectiYe to prolong li~e or cure a patient.

WO 95~11977 ~ ¦ 7 ~ ~ 4 3 PCT/US94/12382 It is apparent therefore that the present invention .~ ; r~
çr7mrociti~7nc~ çr7mhin~ti,7n~ and methods for treating proliferative and infectious disease wherein a cell possesses a cell surface receptor associated with the disease state. For example, the invention includes pl,Gl ,l,a..euli~Gl c.7" 1~ for use in the treatment of 5 human carcinomas, malaria, ~ylJ~ 1l7~ .1,1:r~;~, inflommot~7ry diseases and .y. The cr.1 l ,l..7~:1;. .11 can contain an antibody, or ligand for the antigenspecific to the disease state, conjugated to bryodin 2 of the present invention. The c~... llll ,~:~;. ." can also include other ligands conjugated to bryodin 2 0r other toxins, .. 1,~ .. 11.~.~1~.. 1,~ agents, drugs, enzymes, etc.
o The toxin-ligand and fusion molecule cr7mrnciti~7n~ of the invention can besdl~ lel cd using CU~ Liu~al modes of administration, including but not limited to, illL~G~ uu~ G~e.iLu..~,al, oral, ill~lalyl~ GIi~. or ~ directly into the site ofdisease. IIILIG~,IIOU~ ~Illll;ll ~.lnl;,7~l is preferred.
The cull~,uu~iLhJll~ ûf the invention can be in a variety of dosage forms which include, but are not limited to, liquid solutions or suspension, tablets, pills, powders, 7~ 1 (11 1. ~, polymeric ~llh,~ucG~J~ul~ or ll.l.,l u . .,~h,l~, liposomes and injectable or - . infusible solutions. The preferred form depends upon the mode of alll.;l.; ,LI4~iu.l and the therapeutic arFlic~tion The r.~7. ~ o~:l t71-c ûf the invention also preferably include uu~ LiGIlGl rl -- ",~ y acceptable carriers and adjuvants known in the art such as human serum albumin, ion ~Yrhon~ers~ alumina, lecithin, buffer substances such as rhn~r~"t~, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate.
The most effective mûde of ad~u~ d~iull and dosage regimen for the CullluOa;LiOI~S of this invention depends upon the severity and course of the disease, the patient's health and response to treatment and the judgment of the treating physician.
Accordingly, the dosages ofthe ~.f.1lll.~7~ ,., should be titrated to the individual patient.
N~,~ .,. Ll.~ , an effective dose of the ct7mrn~iti~7n~ of this invention can be in the range of from about 1 to about 2ûûû mg/m2.

. = .. .. _ . = .

WO95/11977 2 1 7 ~ 9 ~ 3 PcT/uss4n23s2 The inter-lulaL;ullalli~- of dosages for an~mals of various sizes and species ând humans based on mg/m2 of surface area is described by Freireich et al., 1966, Cancer Chemolher. Rep. 50:219-244. ~ tm~ontc in the dosage may be made to optimize the tumor cell growth inhibiting and killing response, doses may be divided and a.ll.l;lf.a~.cd s on a daily basis or the dose reduced ~JIUpOI ~iO~ depending upon the situation. It wûuld be clear that the dose of the ~ of the invention required to achieve the desiredeffect may be further reduced with schedule u~ ;n.~
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for o illustrative purposes only and are not to be construed as limiting the scope of this invention in any manner.
EYample 1 Pu. ' ~ t' of Bryodin 2 from Bryonia dioica This example describes the ~ Liull of total protein from the root of Bryonia dioica and the separation of the ribosome-ill~.,LivL~ proteins, including the novel protein bryodin 2.
Bryonia dioica roots (Poyntzfield Herb Nursery, Ross-shire, Scotland) were 20 cleaned, peeled, shred and l.. ",.ng. ..1;~. 1 using a Waring blender in phosphate-buffered saline (PBS, 1 liter PBS :55ûg root material). The slurry obtained was stirred for 16 hours at 4C and strained through cll~s~clùL~ er removal of the plant material, the filtrate was centrifuged at 15 xg for 15 minutes at 4C to remove large particles and then centrifuged a second time at 50 xg for 20 minutes to clarify. The aU~.llL~LII~ was then 2s filtered through a sterile 0.22 micron filter and dialyzed versus 5 mM sodium phosphate buffer, pH 6.5.
Plant proteins were then separated on the basis of their charge and size using afive-step procedure. First, the dialyzed root extract was applied to a CM-Sepharose column (Pharmacia, Uppsala, Sweden), , ' ' ~ to 5 mM sodium phosphate pH 6.5.

WO 9~/11977 '21 7 ~ 9 4 3 PCT/US94112382 Proteins were eluted from the column using a salt gradient of 0 to 0.3 M NaCI. Second, 4 ml fractions were collected and the optical density of the efiluent was monitored at 280 nm (Figure 1). The chromatography fractions were then evaluated by ele~,L.upl.vl~...is.
Fifteen )11 aliquots of each collected fraction were added to SDS-PAGE sample buffer, boiled at 100C for 5 min. and separated on 4-12% SDS-PAGE gradient gels (Laemmili, 1970, Nature 227:680-685). The gels were then stained with Coomassie blue to resolve the separated proteins (Figure 2).
In the third step of the purification, fractions 9 through 15 which contained a 27 kDa protein band were pooled and then cu"ce~ ed to a volume of less than 8 mlo using a Centriprep 10 (Amicon, Bedford, MA). The fourth step was to apply the r to a S;~ ;VII column TSK-3ûO0 (TosoEIaas, Inc., Philadelphia, PA) and then to elute the plant proteins isocratically. Three ml fractions were collected and the êluate was monitored at 280 nm (Figure 3) . Following sizc-cA~.lu.,;v.l ~,lu~ ,"._lr,~
the fifth step in the purification process was to analyze the fractions by SDS-PAGE as described above, except that a 12% SDS-PAGE gel was used. Proteins were resolved by Coomassie blue staining. Two discrete protein bands migrating at 29 kDa and 27 kDa were observed in the peak fractions 58 through 64 (Figure 4). These fractions were pooled separately and this material was used for further ..l,~ r~
E~mple 2 Amino Acid C`r . ~ Orl3~yOdin 2 In this example, the amino acid çr7~rn~itir7~ of the protein comprising the 27 kDa band designated bryodin 2 is determined and compared to the amino acid ~ 71~ of bryodin and bryodin-L. ~mino acid analysis of electroblotted bryodin 2 was performed with the model 420A d~ liVd~;~.GI/ ~GI (Applied Biosystems, Inc.) after manual vapor phase hydrolysis with 6N HCI at 165C for I hr (Dupont et al., 1989 in Hugli, T.E., ed., Techniques in Protein Chemistry, pp. 284-294, Academic Press, Inc., San Diego, CA).

wo 95/11977 . 2 t 7 4 9 4 3 PCT/US94/12382 From this analysis it appears that bryodin 2 is a novel bryodin ribosome-inactivating protein ~ llu~ulLly different from bryodin and bryodin-L.
Table 1 bryodin 1I bryodinLI bryodin2 du.,.~/lllol) (Its-lu~ l-OI) (It~ U~3/11101) Lys 8,6 10.8 0.4 ~[is 1.9 1.0 abs Arg 11.8 11.0 8.5 AsY 22.5 25.5 14.0 Thr 15.1 17.4 13.1 Ser 30.2 24.4 6.5 Glx 17.7 18.9 33.2 Pro 6.7 7.2 lS.0 Gly 11.4 11.4 16.1 Ala 22.4 24.1 28.7 ~/2Cys 0.24 abs2 abs V~l 15.6 14.4 34.2 Met 1,6 2,2 abs lle 15.1 15.4 23.3 Leu 28,3 24,5 28.3 Tyr 14.2 11,7 . 5.0 Phe 8,3 7,4 18.5 Trp 2.0 abs ND3 1, Values for amino acid residues taken from European Publication Number EPO 39004L~, 2. abs means the amino acid residue was either not present or was present in a~nounts below odetection.

3. ND, not determined.
Example 3 N-Terminal Amino Acid Sequence Analysis of Bryodin In this example, the N-terminal amino acid sequence of the 27 kDa and 29 kDa proteins contained in the pooled fractions was dPt~rminP~ The first 32 amino acid residues of the 27 kDa and 29 kDa protein bands were ~ ,"~ l y dPt~in~ The protein comprising the 29 kDa band was found to be identical to the bryodin (bryodin 1) 20 described by Stirpe. The protein comprising the 27 kDa band was found to have an WO9S/11977 ~ ~3 PCT/US94/12382 N-terminal amino acid sequence substantially different from the N-terminal sequence of bryodin I (Figure 5). We have designated the novel toxin bryodin 2.
N-terminal amino acid sequences were determined by using the following methods which are briefly described. The protein bands were i~ lividu~llly recovered from 5 SDS-polyacrylamide gels by clc.,LI ubluLLillg onto a Problott membrane (Applied Biosystems, Foster City, CA) using a Mini-transblot EIC~L~ U~I~OI e~i~. Transfer Cell (Bio Rad Laboratories, Richmond, CA) (M~tq l,' d, 1987, J. BioL Chem. 262:
lû035-10038). The membrane was stained with Coomassie brilliant blue, then destained, and the 29- and 27-kDa bands were excised for subsequent amino terminal sequenceo analysis.
Samples were sequenced in a pulsed liquid phase protein sequencer (Model 476A, Applied Biosystems) equipped with a vertical cross-flow reaction cartridge using.Lul~l's released cycle programs. ~h~ yl~lliùllydall~ulll amino acid derivatives were analyzed by reversed-phase HPLC with a PTH C18 column (Applied Biosystems) using15 sodium acetatelLeL~ d. urul a/acetonitrile gradient for elution (Tempst and Reviere, 1989, ~InaL Biochem. 183:290-300). Data reduction and ,~ "." were performed using a Model 610A UIIIUIII~LUgI~IIII analysis softwâre (Applied Biosystems).
The amino-terminal amino acid sequence of BD2 was performed with 47 pmoles (based on the initial yield of identified Val-1), electroblotted onto Problott membrane. A
single amino acid sequence was obtained and ~ .,u.~ ;." of PTH-amino acid derivatives was possible up to residue 32 (Figure 5; Seq. I.D. #1).
l~xâmple 4 Determinâtion of the Amino Acid Sequence of Peptide Frâgments of Bryodin 2 In this example, the 27 kDa protein (BD2) isolated by PAGE was cleaved into fragments using cyanogen bromide and various proteinases. The peptide fragments were isolated and the amino acid sequence of certain fragments df tPnninf~d The obtained WO 95/11977 PCT/US9~/12382 ~ ~174~3 amino acid sequences were analyzed for overlaps and homology with known ribosome-,.Liv~Lill~proteins.
BD2 was cleaved with cyanogen bromide by dissolving BD2 into 30 1ll of 70%
formic acid and adding enough cyanogen bromide (30 mg/100 lal) in 70% formic acid to provide a l,000-fold molar excess over methionine The reaction was allowed to proceed under a nitrogen cushion for 4 hours at 30C and for an additional 18 hours at 22C in the dark. The cyanogen bromide peptides were separated by gel permeation cll~ un~L~ hJ
using a 600 x 7.5 mm Bio-Sil TSK-250 column (Bio-Rad Laboratories, Richmond, CA)c~luil;bl ~Lell in 0.1% TFA containing 40% acetonitrile at a flow rate of 250 ~LUmin. The lo eluent was monitored at 280 nm and peaks were collected manually for further analysis.
Purified BD2 or purified cyanogen bromide peptides derived from BD2 were cleaved with the proteinases trypsin (I~(tosylamido-2-phenyl) ethy H,l~lulull~,;llyl ketone-treated, Worthington), Lys-C and Glu-C (Staphylococcus aureus, Boehringer M~nnh~im) Protease cleavages were done in 40 111 0.1 M Tris-acetic acid buffer containing 2 M urea, pH 8.5, at 37C for 16 hours. The enzyme substrate ratio was I to 25. The enzymatic digests were acidified with 10% TFA to pH 2 and separated by reversed phase HPLC.
Reversed phase HPLC was carried out using a 2.1 x 100 mm RP-300 cartridge column (Applied Biosystems) and a I x 100 mm Cl 8 Vydac column (The Nest Group) at a flow rate of 100 Ill/min and 40 Ill/min, I~ 'y, at 40C. Linear acetonitrile gradients from solvent A (0.1% TFA in water) to solvent B (0.09% TFA in acetonitrile) were used for elution. The eluent was monitored at 215 nm and peaks were collected manually.
Peptides were sequenced on polybrene-coated glass fiber discs (Applied Biosystems). Automated sequence analysis was performed in a pulsed-liquid protein sequencer model 476A (Applied Biosystems) using manufacturer-released cycle programs.
PTH-amino acid derivatives were analyzed by reversed-phase HPLC with a PTE~ Cl 8column (Applied Biosystems) using a sodium acetate/teL~ yJ~ruld~ cc~o-lil-ilc gradient for elution. Data reduction and qll~ntit~tirn were performed on a Macintosh IIsi computer wo gs/ll977 2 ~ 7 ,~ ~ 4 3 PCT/U~94/lU82 (Apple Computer, Inc.) and model 610A chromatogram analysis software (Applied Biosystems).
Edman d~. ~daliul~ of selected fragments derived from BD2 by cleavage with trypsin, cyanogen bromide and further digestion with Lys-C and Glu-C protease are shown 5 inFigure 6. Cleavage of BD2 with cyanogen bromide generated two major peptides (M2 and M4). Peptide M2 (MR=14,000) was the ~ lr~ cyanogen bromide peptide of BD2 (Seq. I.D. #I) and is shown in Figure 6. Peptide M4 (MR=12,000) repre3ents the ci~bw-ylL~ dl cyanogen bromide peptide of BD2 The ~ lot~ dl amino acid sequence of M4 is shown in Figure 6 (Seq. I.D. #4).
lo Digestion of M4 with Lys-C protease generated three major fragments. The amino terminal sequences of two of those fragments, designated M4/K2 (Seq. I.D. #S) and M4/K11 (Seq. I.D. #6), are shown in Figure 6. Peptide M4/K11 was 5~ ~ ,r".~ with Glu-C protease, generating four fragments. Peptide E4 (Seq. I.D. #7) provided overlap ;,,r,,,,"~,;"" and.extended the sequence of M4/K11 by 14 residues.
Peptide M4, preceded by a methionine residue, provided an overlap with peptides T21 (Seq. I.D. ~3) and K2 (Seq. I.D. #5), extending the sequence of M4 by 25 residues.
BD2 belongs to a family of plant ribosome-;ll~.~,Li~GLi..g proteins, including momordin, shiga toxin a-chain, shiga-like toxins I and Il, and ricin A-chain. A comparison of the amino acid sequences of BD2 and momordin II is shown as an example in Figure 7.
20 Peptides T10 (Seq. I.D. #2) and M4/K11 (Seq. I.D. #6) were aligned with the momordin sequence (Seq. I.D. #13) based on similarity, without providing overlap information with T21 and M4/K2, }espectively. BD2 shared with momordin 77 amino acid residues out of 157 ~,u~ vl~ (4g.0% identity).

wo 95111977 2 1 7 4 ~ 4 3 PCT/US9~/12382 Example 5 Dct~ ;o~ of Protein Synthesis Inhibition Activity In this example, the ability of BD2 to inhibit protein synthesis was determined in a cell-free rabbit reticulolysate trans~ation system. Bryodin 2 was found to be a very efficient inhibitor of protein synthesis, having activity similar to that of bryodin 1 and substantially more active than gelonin or ricin A chain.
Protein synthesis inhibition activity was determined using a cell-free rabbit reticulocyte Iysate translation system (Promega Biotec, Madison, WI). The assay was 0 performed as per the Illclllrc~,~Ulel'b instructions. Briefly, toxin proteins were mixed in a volume of 25 1ll per reaction with rabbit reticulocyte Iysate (70% of reaction volume), a mixture of all amino acids (minus leucine) at 1 nM, RNasin rihnn11rl~c~ inhibitor (20~), 0.5 mCi/ml [3H]-leucine, and RNA substrate (0.5 llg). The reaction was incubated at 30C for 5 minutes and terminated by adding 1 M NaOH with 2% H2O2. The translation product was ~ cLed using ice-cold 25% Lli~J,occeLic acid (TCA) with 2% casamino acids on ice for 30 minutes. The radiolabeled proteins were harvested on glass fiber filters, rinsed with cold 5% TCA, rinsed with acetone and then dried. The amount of protein translated was quantitated using a s~-in~ tinn counter.
Both isolated protein toxins bryodin 1 and bryodin 2 were found to be potent irlhibitors of protein synthesis with ~Cso values of 0.007 and 0.017 nM, I~l,e~ ,ly. In the salne assay, gelonin, a type I ribosome-inhibiting protein, was found to have an EC50 value of 0.049 nM and ricin ~ chain was found to have an ECso value of 0.05g nM.

~ 7 49 43 ~
E~ample 6 Isolation of M ~ l Antibodies Specific for Bryodin 2 In this example, murine monoclonal antibodies are generated specific for br~odin 2.
Antibodies were generated which were not cross-reactive with bryodin I or with control protein toxins ricin A chain, momordin or gelonin.
Briefly, four-to-six-week-old female BALB/c mice were initially immunized with two ~ h.~. u~ injections (0.1 ml) and one i~ uilcdl injection (0.2 ml) of 2 50:50 mixture of purified BD proteins (BDI and BD2; 200 ~Lg total protein) and Ribi adjuvant 0 with ISA 50 Seppic Oil (Ribi Immlln~lrh~mi~l, Hamilton, MT), followed by a 0.3 ml ;IlLl~r~ UlleGI injection of BD protein, 60 llg, in ISA ~0 Seppic Oil, on week four.
Another 0.3 ml intraperitoneal injection of 60 llg BD protein was given on week seven to boost imm~lni7~firm Spleen cells from an immunized mouse were removed three.days after the final imml ~ni7~ficm and fused with the myeloma Ag8.653 at a ratio of 3:1 with 40%
pul~,Llly~ glycol 1450. The fused mixture was plated in HAT (I~ u~ ll;llc-d..lillu~ lill-thymidine) medium with dy~Jl w-illlaLcly 2 x 106 ~ u~t~,Jl~ll (BALB/c) at û.2 ml/well into 10 96-well plates. Hybridomas secreting antibodies specific for BD2 were selected by ELISA using plates coated with BD2. Briefly, Immulon II plates (Dynatech, Chantilly, VA) were coated with 0.3 llglml BD I or BD2 overnight at 4C in 0.1 mllwell carbonate buffer (0.1 M sodium carbonate/sodium ~ OIbUI~ ., pH 9.6). Plates werewashed with phosphate-buffered saline (PBS), blocked with 200 IlUwell Specimen Diluent (Genetic Systems Corp., Redmond, WA) for 2 hours at 4C, and rewashed with PBS.
Sample ~u~ L.~lL from wells containing growing clones and Specimen Diluent (0.05 ml each) were added to each well, incubated at 4C for 2 hours, and washed three times in PBS. Goat anti-mouse horseradish peroxidase (HRP) (0.1 mVwell), used at 1:3,000 dilution in Conjugate Diluent (Genetic Systems Cûrp.), was incubated for 1 hour at room Lc~ UI C and washed four times before addition of 0.1 ml/well substrate (tetramethyl ben_edine in substrate buffer, Genetic Systems) and further incubated for 10 minutes. The WO95111977 2 1 7 ~ ~4 ~ PCT/US94/12382 reaction was stopped with 0.1 mVwell 1.3 M ~2SO4 and the optical density quantified at 450 nm on a Bio-Tek microplate reader (Winooski, VT).
Hybridomas secreting anti-BD2 antibodies were selected and cloned by two rounds of limiting dilution and retested for reactivity by ELISA as described above. Limiting 5 dilutionswerecarriedoutinIMDM,10%fetalcalfserum,1%penicill;~1~Ll~uLu...J.,;,l.Two BD2-reactive antibodies were selected and purified from culture ~ by affinity ~ ,,y using Gamma Bind Plus (Pharmacia). Protein ~ was determined by OD280 Specificity assays for the two selected anti-BD2 antibodies were performed using0 the ELISA assay described above with BD2 except that, in addition, assays using ricin A
chain or gelonin were coated onto the ELISA plate at 0.3 llg/m'.. Detection of bound antibody was done with goat anti-mouse IgGl-HRP at 1:1000 dilution (Southerr.
Rirt~rhn~ gy, Birmingham, AL). As shown in Figure 8, ., .r,llr rlr", -~ antibody 50-44-3 recognized BD2, but not BDI or ricin A chain. The slight reactivity with BD1 is most 15 likely attributable to a small amount of BD1 i ,l tA~ . .., present in the BD2 ,~l~iiliiL'u--.
Additionally, 50-44-3 did not react or recognize MMC or gelonin (data not shown). A
second antibody, 50-43-1, was also isolated which has a specificity similar to 50-44-3 (data not shown). This second antibody appears only to differ in having a lower affinity ûr t ~tdity t`or BD~

WO 9~/11977 , PCT/US94/12382 ! 43 Exnmple 7 ToYicity of Bryodin 2 in vivo In this example, the single dose LDso was determined for bryodin 2 in mice. It was determined that a dose of 8 mg/kg ;IILIap~ u~ ally or greater than 10 mgfkg aJ~Iilfi~ d illLld~ .vu~ly was sufficient to kill half the mice tested. The single dose LDso for bryodin is reported to be 14.5 mg/kg when administered i~ u~ lly.
Briefly, toxicity was determined by both intraperitoneal and intravenous (via the tail vein) injection. The purified toxin was diluted in phosphate buffered saline to reach 0 final adl~ullia~ d doses of 3 to 20 mg/kg. Mice (type) were placed in groups of 2-4 and a~,lu..,;,L~l~d a quantity oftoxin. Animals were monitored for at least 14 days following injection of toxin. For UVIII~ e necropsy analysisl animals were ;IILl av ~..luui~lJ
injected with 20 mg/kg toxin, sacrificed after 24 hours, and selected tissues were analyzed using gross and IIUW ua~,Op;C techniques.
Bryodin 2 was determined to be slightly more toxic to mice than bryodin I when a.ll,li..;.,L~,~d ;llLla,u~,l;Lull-,a'ly and when administered ;llLld~,.-VU~ly (Table 2).
C~ a; v ~ l u~u~Jy determined that liver toxicity was the cause of death in animals receiving a lethal dose oftoxin. ~i~lo~h~mir~l analysis oftissue from injected animals showed liver lesions. Additionally, SGOT and SGPT were elevated in these animals.

WO 9Stll977 . 2 1 7 4 ~ {~ 3 PCT/US94/12382 Table 2 Lethal Toxicity of BDI and BD2 to Mice lRIP Route Dose # Mice % Survival (mg/kg) BD2i.v. 5 4 100%
i.v. 6 4 100%
i.v. 7 4 100%
i.V. 8 4 75%
i.v. 10 4 75%
i.v. 12 2 0%
i.v. 14 2 0%
BDIiv. 12 4 100%
i.v. 12 4 100%
i.v. 16 2 100%
i.v. 18 2 100%
i.v. 20 2 100%
BD2i.p. 7 4 100%
i.p. 8 6 50%
i.p. 10 2 0%
i.p. 12 2 0%
BD1i.p. 10 4 100%
i.p. 12 2 100%
i.p. 14 2 100%
i.p. 16 2 100%
ADimals (20-25 g) were observed ~or > 14 days îollowing injection. BD RIP was diluted in PBS prior to injection.

_, . , . . . . _ _ . . . _ , _ _ _ WO 95111977 , PCTIUS9~/12382 ?174943 ~
Example 8 Chemical Conjugdtion orBryodin 2 to Form an l In this example, bryodin 2 was covalently crosslinked (or conjugated) to a chimeric mnnnnlnn~l antibody imml~nnlnejr~lly reactive with a highly specific tumor-associated antigen, chimeric BR96 ~ATCC HB 10460). The antibody was intended to direct the ribosome~ iv~ g protein to the target tumor cell and to protect the patient from the inherent toxicity of the RIP. Activity of the; " " "" ", .l . .~; " was determined by testing the abiiity ofthe i"""~ .lu~ to bind antigen on membranes isolated from a breast carcinoma cell line and a d~.rl Ill;lIAI;I~I~ ofthe ability ofthe ;l,l,..,~ rl~; to kill the same cell line.
Partially purified; " ,. ", l ". .~ ; " has been shown to bind to the membranes of a human breast carcinoma cell line (H3396) known to be BR96 positive and to be toxic to these ceiis.
Chimeric BR96 (15.6 mglml) was thiolated with the addition 4f a three-fold molarexcess of 2-rminnthinl mf . (2-IT, Pierce Chemical Company, Rocicford, :L) in 0.2 M
sodium phosphate buffer (pH 8.0), I mM EDTA for I hour at 37C. Unreacted 2-IT was removed by ulllullla~u~ld~ y through a PD-10 column (Pharmacia). BD2 (4.6 mg/mi) was derivatized with a three-fold molar excess of buc~il,il"i iylu~.,~,l,u~yl~ methyl-o (2-~ ~ i iylLiiLllio)-toluene (SMPT) in 0.2 M sodium phosphate buffer, pH 8.0, I mM EDTA at room lelll~,.dlul~ for 60 minutes followed by ~ lur~ LO~ y on aPD-10 column. Themodified toxin and thiolated antibody were mixed in a 5:1 molar ratio and incubated at room Ltl-l,uL"dLul~: for 16 hours to allow disulfide bond formation.
T"""",,..l..~;" conjugates were applied to a TSK-3000 ,i~,-h,u,lu ,;vl, column and 25 separatedfromfreetoxin. The;"..."l..,.l.,~;..havingamolecularweightofaboutl80kDa and free antibody having a molecular weight of about 150 kDa eluted together and were further purified by chromatography on Blue-Sepharose (Pharmacia) (Figure 9A). Prior to adsorption to the Blue-Sepharose, the partially purified ;l."""""~ " sample was dialyzed into 0.1 M sodium phosphate, pH 7Ø The Blue-Sepharose was r . "' dL~d with the wo 95/11977 ~ l 7 ~ 9 4 3 P~JUS94II2382 same buffer and the dialyzed immlln~tm~in sample was batch adsorbed to the Blue-Sepharose (5 ml resin/5 mg ;""",l.,.,l..~;") for 16 hours at 4C. The mixture of Blue-Sepharose and imrnunotoxin sample was packed into a 5 ml Econo column (Bio-Rad, Richmond, CA) and 1 ml fractions were collected as the column was eluted with a two-step gradient of increasing NaCI I~UI~,Gllll _L;UI-~ in 0.1 M sodium phosphate, pH 7Ø The two steps of the gradient were 400 mM NaCI followed by 800 nlM NaCI (Figure 9B).Quantitation ofthe amount of immllnntnyin in each fraction was determined at D280 and analyzed by non-reducing SDS-PAGE analysis ~igure 9C).
Antigen binding activity of chiBR96-BD2 and chiBR96-BD 1 illllllUlnJ~C/~ill lo conjugates was determined by measuring the binding of the ;~ conjugates to isolated Ill~lllbl ~ 5 of the human breast carcinoma cell line H3396. Both chiBR96-BD2 and chiBR96-BD1 conjugates were found to bind antigen on H3396 membrane similarly.
The conjugate may bind slightly better than " ". . ,";. I~A I r~ BR96 antibody (Figure 10), the increased binding possibly being due to the presence of antibody aggregates formed during the .m j~gAl;. ", procedure. It has previously been shown that increased binding activity has been associated with dimers of BR96 (Wolffet al., 1993, CancerI~es. ~3:2560). Both BD2 and BD I ~nrnnill~:~tpd to antibody show no detectable bindirlg to H3396 membranes (Figure 10).
.,s used for binding studies were from H3396 cells prepared by c-,-l~,iru~illg 5 x 107 cells at 1500 xg for five minutes and frozen at -70C. The cell pellet was thawed at room I~IIIIJ.,. _Lul c: and Iysed in 10 niM Tris-HCI, pH 7.4, 5 mM EDTA, 0.5 mM ph~,.l.rllll~L~yl ~ulr~llylnuol;dc (PMSF) at 4C for 15 minutes, and ll.. n~;~ .,:.. ;i The Iysed cells were centrifuged at 1500 xg at 4C for 5 minutes to clarify the `"1~ llAlA l' The ~ was further centrifuged at 7500 xg at 4C for 80 minutes and the pellet 25 was . ~ J in PBS, 0.5 mM PMSF, 25 mM indo~PtAmi-lP ~ ...,;. were collected by ~....l .. l~ l~,...g the solution at 7500 xg at 4C for 80 minutes and the pellet ,t . ..1~1 in PBS, 0.5 mM PMSF, 25 mM in~ p~ and the protein determined by absorbence of A280 21 7~9~3 WO 95/11977 , PCT/I~S94112382 o Assay of binding to the membrane was carried out by coating the su&ce of Immulon II 96 well plates (Dynatech Labs, Chantilly, V~) with 10 ,ug/ml H3396 membranes in 0.1 M sodium carbonated sodium bicarbonate buffer, pH 9.6, for 16 hours at 4C. The plates were blocked with specimen diluent (Genetic Systems Corp., Seattle, 5 WA) for one hour at room lt.~ r ld~UI~ and incubated with ;.."l.""~ ". at 4C for 16 hours. Plates were washed with PB S three times followed by the addition of goat anti-human (heavy and light chains) horseradish peroxidase (American Qualex, La Mirada, CA) at 1:1000 in conjugate diluent (Genetic Systems Corp.). After incubation for one hour at room t~lllLJ~"dlUI ~, plates were washed five times with PBS, and developed with0 tetramethyl benzedine chromagen (Genetic Systems Corp.) for 10 minutes. The reaction was stopped with 1.3 M H2SO4 and, " ." " " ,",~ was quantitated using a Bio-Tek microplate reader (Winooski, VT) at 450-630 nm.
Cytotoxicity of the chiBR96-BD2 ;" " ~ ,. was determined by plating H3396 tumor cells onto 96-well nat bottomed tissue culture plates (1 x 104 cells/well) and kept at 5 37C for l6 hours. Dilutions of;,,,,,.,l,,, l~ .. or imm.-nr)toYin cu.,l~,o..~ b were made in culture media (IMDM, 10% FBS, 1% Peniclllin/~L~ .l.,y~,;..) and 0.1 ml added to each well for 96 hours at 37C. Each dilution was done in triplicate. ~er incubation with the ;., . " ,, . "r ,l r-~, ", or toxin ~ ~ , the wells were washed twice with PBS and 200 ,ul/well of 1.5 IlM calcein-AM Q\~olecular Probes, Inc., Eugene, OR) was added for 20 40 minutes at room ~ ,.dLul~. Following incubation with calcein-AM, the amount of nuOl ~ was determined using a FIUOI ~a~ r- Cu.~ t- dL;~ Analyzer ~axter Healthcare Corp., Mundelein, IL) at ~AI,iLd~h)l~ ;Oll ~va~ of 485 nm/530 nm.
The data are presented as percent cell killing for each treatment calculated as:

WO 95111977 . PCT/U594tl2382 2 1 7¢ 9~3 (sample signal-background signal) 100- x 100 (maximal signal-b,.,h~.uul~d signal) Background signal was measured from cells treated with Triton X-100 and maximal signal was measured from nûn-imn~llnntoxin treated cells.
Cell killing activity of chiBR96-BD2 and chiBR96-BDl illUllUl~UtU~ ill conjugates 10 were found to be cytotoxic to H3396 cells at a similar level with an ECso=100 pM
(Figure llA). H3719 colon carcinoma cells which express ~"lri~ hl,l. . Ievels of BR96 antigen were found to be relativeiy insensitive to both ehiBR96-BD2 and chiBR96-BD1 (ECso > 5 x 104 pM, Figure 1 IB).
Protein synthesis inhibition activity was determined by measuring [3H]-leueine 15 ;..~.UIIJUI~I~iOll into cellular proteins following a 20-hour incubation of ;"...,.,... ~..1; with H3396 cells and a four-hour pulse with ~3H]-leucine. The; ~ t~ chiBR96-BD2 and ehiBR96-BD1 were added to H3396 eells (1 x 104 eells/well) in a 96-well mierotiter plate. The cells were grown to 75% eon'duenee in IMDM medium with 10% FBS. The eells were ineubated with the test material for a total of 24 hours, the last four hours with 20 1 ~ICi of [3H'-leucine added to each well. The cells were Iysed by freeze-thawing and harvested using a TomTec Cell Harvester (TomTee Ine., Orange, CT). Ill~,ul~ùl~iul~ of [3H'i-leueine into cellular protein was determined by an LKB Beta-Plate Liquid S~intill~tinn Counter.
Example 9 Cloning of Bryodin 2 from the Le~ves of Bryonia dioica In this example, degenerate nli¢~- ~, ."~ 1 itlc probes were used to isolate a small region of DNA, amplified from 3~yo~nia dio;ca mRNA, that ~,ull.,~l~olldGd to an amino 30 aeid sequence of Bryodin Z. These regions of DNA were sequenced and a series of wo 95111977 '2. l 7 ~ ~ 4 3 PCTIUS94/12382 .1;, v~uulcvLide primers exactly corresponding to the determined DNA sequence were designed and, together with degenerate primers designed from the amino acid sequence of internal peptide fragments of BD2, were used to amplify a longer stretch of DNA encoding BD2. Having auuc~arully isolated and sequenced a substantial portion of BD2 DNA, 5' and 3' RACE techniques were used to identify the exact 5' and 3' ends ofthe cDNA
sequence encoding the entire Bryodin 2 open reading frame.
Briefly, total RNA was extracted from Bryonia dioica leaf material by finely grinding leaves in dry ice and hflmfl~f ni7in~ in TRI Reagent (phenol, guanidine LLIv~ a~ Molecular Research Center, Inc., Cincinnati, OH) at 10 mUg tissue. RNA
0 was cxtracted with chloroform, ~ u;~;LdL~d with isv~l u~Jdllvl, washed with 75% ethanol and dissolved in DEPC (diethyl pyrocarbonate) treated water. Total RNA was quantitated and analyzed by elc~,L,u~,~,vl~a;, in rul."àl~c~.ylc-agarose gels and visualized by staining with ethidium bromide.
First strand cDNA was synthesized by incubating 1 ,ug total B. dioica leafRNA
template and 10 pmole oligo(dT)-primer XSCT17 (Table 3) at 65C for 10 min. and then on ice for 2 min. to allow annealing to occur. This was followed by adding synthesis buffer (20 mM Tris-HCI, pH S.3, 50 mM KCI, 2.5 mM MgCI), 10 mM dNTP mix (5 llM
final concentration), 10 mM dithiotreitol and 200 U Superscript reverse ~ f to the RNA rnixture and incubating for 30 min. at 42C. RNAse H (2 U) was then added and the rnixture was incubated for an additional 10 min. The cDNA synthesized in this reactionwasPCRamplifiedusingtwosetsofdegenerate-~lig-..,.,-.l~^vL;-if~(a)BD2pl4 (12~-fold degeneracy) and BD2 pl9, and (b) BD2 pl8 (512-fold degeneracy) and BD2pl9 (See Table 3) at 25 pmole each. An a~lJIu~dlllfll~ly 500 base pair single band was obtained using primer set (b) after separation of the cDNA by agarose gel elc~L,u~ ol~a;S
25 and ~;..Udl;~L;UII with ethidium bromide.
... . .. _ . .. .... , .. , .. _ . _ . , ... . . . . ..... .. . . . .... . . . _ . . _ . _ ... ..... .

Wos~ 977 2 ~ 7~43 PCT/US94/12382 Table 3 BD2 Ol;6J~ otides Used for Cloning BD2pl4 5'-ACN TAC(I~ AAA(G) ACN TTC(I) AT-3' (Seq. I.D.#16) 5' oligo, (14-20 aa) TYKTFI 128-fold BD2P 18 5 '-GGN GCN ACN TAT AAA(G) ACN AT-3 ' (Seq. I.D.#17) 5' oligo (12-20 aa) GATYKTFI 512-fold BD2p 19 5'-CTC A(G)AT ATA C(l )TT A(G)AA T(C)CT CGC AGC CTC-3' (Seq. I.D.#18) 3' oligo (163-169 aa) EAARFKYI
XSCT17 5'-GACTCGAGTCGACATCGAl1111111111111111-3' (Seq. I.D.#l9) 3' oligo XSC 5'- GAC TCG AGT CGA CAT CG-3' (Seq. I.D.#20) 3' oligo BD2 3'RACE#2 5'- ACC ACA CTC ACG GTT GGA ACT CCA-3' (Seq. I.D.#21) 5' oligo (24-31 aa) TKLTVGTP
BD2 5' RACE#4 5'- TGG AGT TCC AAC CGT GAG TGT GGT-3' (Seq. I.D.#22) 3' oligo (24-31 aa) TKLTVGTP
BD2 5tRAcE#5 5'- C GTT CAC TAC ATC TTA AGC CAC AGT GAC-3' (Seq. I.D.#23) 3' oligo (62-71 aa) VTVALDVVNV
BD2-3'RACE#11 5'- GA CTT CCT TAT GGA GGG AAT TAC GAT GGC CTT-3' (Seq. I.D.# 24) 3' oligo, (104-114 aa) RLPYGGNYDGL
5' RACE AP 5'- GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3' (Seq. I.D.# 25) M13 P~everse Pr~mer 5'- CAG GAA ACA GCT ATG AC-3' (Seq. I.D. #26) M13 Forward Primer 5'- CTG GCC GTC GTT TTA C-3' (Seq. ID. #27) The products of the PCR reaction were subcloned into the vector pCRlI
5 (Invitrogen Corp.) using the TA cloning kit. Briefly, PCR product was combined with ligation buffer, pCRII vector (50 ng) and 4 U T4 DNA ligase at l ~ 3 and l :5 vector to insert ratios. The reactions were incubated overnight at 16C. DH5a E. coli were WO 95/119M , PCT/US94/12382 ~ 4q 43 Llal. .ro~ d with 3 ul of the ligation reaction mixture by incubation on ice for 30 min., followed by a 42C incubation for 45 sec., 2 min. on ice, and an incubation at 37C in 450 1ll of SOC (20 nlM glucose, 10 mM MgSO4, 10 mM MgCI2, 2% tryptone, 0.5% yeast extract, 10 mM NaCI, 2.5 mM KCI) for l hr. Cells were plated on LB agar plates containing ampicillin (50 llg/ml), 25 ~d X-Gal (40 mg/ml in dimethyl formamide) and 25 ,ul isopropyl l-thio~ D-galact~.~.ylailos;~t (IPTG, 240 mg/ml) and incubated overnight at 37C.
PCR analysis of cuv~ ;llall~ clones was carried out using Universal M13 forward and reverse primers (Table 3) followed by ~ ' on by agarose gel CI~ V~
Briefly, positive clones were incubated at 37C in 50 1ll of LB broth containing 50 ,ul/ml ampicillin for 1 hr. Cells (12 ,ul) were diluted in 50 ul of 10 mM Tris-HCI and 1 mM
EDTA and incubated at 95C for 5 min. The PCR reaction (100 ,ul) consisted of lX PCR
buffer (10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 rnM MgCI2), dNTP mix (200 ,uM), 25 pmole each M13 forward and M13 reverse primers, 10 1ll cells (test DNA) and 2.5 U
Taq DNA po~ymerase. Thirty-five cycles were run using a GeneAmp PCR System Model9600 (Perkin Elmer Cetus) using the cycle conditions of 94C for 3 min., 94C 1~ sec., 55C 15 sec., 72C 1.15 min. x 35; 72C 6 min. and hold at 4C. Reaction product was visualized by agarose gel ~ .LIolJl.o.t,;s. Clones were selected for DNA sequencing analysis based on the presence of a 500~ bp insert.
DNA was sequenced by did~ y~u.,lcvLide termination using Sequenase (United States Rio~h~mir~l). Four separate clones containing a nucleotide sequence determined to encode the previously determined amino-terminal amino acid sequence of BD2 were identified. A468bpcDNAfragmentwhichcv--~ vl~d~,dtothemajorityoftheBD2 amino acid sequence was used as template for 3' and 5' RACE techniques, which were WO 9~111977 2 l 7 ~q 9 ~ 3 PCT/US94/12382 used to identify the start codon, to confirrn the amino terminus of the BD2 gene, and to obtain DNA sequence to the polyA tail.
,~mriifi~.Ati~n by 3' RAC~ was carried out by using a 3' RACE System (Gibco BRL). Brie~ly, 0.5 llgtotal BD leafRNAwas incubated with io pmole ~ "....~ ; ir primer XSC-T17 (Table 3) at 65C for 1 min. followed by 2 rnin. on ice. The RNA
rnixture was then mixed and incubated at 42C for 2 min. with synthesis buffer (20 rnM
Tris-HCI, pH 8.4, 50 mM KCI, 2.5 mM MgCI2, 100 llg/ml BSA), dNTP mix (500 IIM
each), and 2 1ll 0.1 MDTT before the addition of 200 units Superscript reverse l"... - ."l,lr~ and a further incubation at 42C for 30 min. The 3' end of BD2 cDNA was 10 PCR amplified with 25 pmoles each of primers BD2 3' RACE #2 and XSC (Table 3) in synthesis buffer with 200 ~M dNTP mix and 2.5 U Taq polymerase. An 800~ bp band was visualized by agarose gel el~.LI UIJhU~ ,is and was cloned into the TA cloning vector pCRII as described previously. Clones containing BD2 sequences were selected by lybrid;~Liull of colonies lifted onto nylon m~mhr~n~-c, as described below, probed with ~32P]-labeled BD2-3' RACE #11.
After incubation, the agar plates were cooled at 4C for 2 hr after which colonies were lifted onto nylon membranes for 1 min. The membranes were incubated in 1.5 M
NaCI, 0.5 M NaOH to denature the DNA followed by neutralization in 1.5 M NaCl, 0.5 M
Tris-HCl, pH 7.2, and 0.1 mM EDTA The membranes were then washed in 2X SSC and 20 the DNA was crosslinked to the membranes in a W Stratalinker 1800 (Stratagene).
P-el.~.,li-l;~dLio.l was carried out by incubating the membranes in 6X SSC, 5XDenhardt's, 0.05% sodium l~yl hllU~I~O~ a~e, 0.5% SDS and 0.02 mg/ml salmon testes DNA at 50C
overnight. Hybridization was carried out with the ~ddioldhcl~d prûbe [32p] BD2 RACE
#11 (0.5 - 2xlO6 cpm/ml) in 6X SSC, IX Denhardt's, 0.05% sodium ~J.1. AI l~pl~ r, and wo gsll 1977 2 ~ 7 ~ ~ 4 3 PCTIUS94112382 100 llglml yeast RNA at ~0C for 4 hr. The membranes were washed with 6X SSC, 0 1%
sodium l~yl~lluplIoa~ te at 37C, followed by exposure to autoradiograph film.
Miniri~cmi~i DNA was made from the positive white colonies and analyzed by PCR and DNA s~qll~nr in~
5' RACE was performed using the 5' RACE System (Gibco BRL) as per manufacturer's illa~l U~iOlls. Briefly, I llg of BD leaf total RNA and 2 pmole primer BD2 5' RACE #S were combined and denatured at 70C for 9 min. followed by incubation at 42C for 30 min. in synthesis buffer 0.01 M DTT, dNTP mix (500 IIM each) and 200 U
Superscript reverse Ll Gll~ )LhaC. in a total volume of 19 lal. The RNA template was lo degraded with 2 U RNase H at 55C for 10 min. cDNA was purified with a Glassmax spin cartridge to remove primers, U..illcu.l,u,~td dNTPs and proteins by adding 95 111 of binding buffer (6 M sodium iodide) to the reaction mix and ~l~llaf;~lillg the reaction contents to a Glassmax spin cartridge. The loaded cartridge was centrifuged at 13,000 xg for I min. and then washed three times in IX Glassmax wash buffer and once in 70%
15 ethanol. The cDNA was eluted with S0 ~LI of water (65C). Purified cDNA was dC-tailed by denaturing I û 1ll of cDNA at 70C for 6 min. This was followed by incubation in IX
synthesis buffer with 2 mM dCTP (20û ~LM) and 10 U TdT in a total volume of 20 111 at 37C for 10 min. and then incubating the mixture at 65C for 15 min.
The dC-tailed cDNA (5 lal) was PCR amplified using 25 pmole each of BD2 S' 20 RACE #4 and 5' RACE AP (Table 3) for 35 cycles as described previously above. DNA
was analyzed by agarose gel CIC~.ll Upl10l ciaiS which revealed various 100-200 bp fragments which were subcloned into pCRlI as described previously. PCR analysis of white colonies using Universal M13 forward and M13 reverse primers identified clones containing inserts.
Two positive clones were selected for DNA sequencing and extended the DNA sequence WO 95/11977 2 1 7 ~ ~ ~ 3 PCTIUS94112382 ~3 for BD2 63 bp upstream from the beginning sequences of the mature BD2 protein. The natural initiating methionine and the putative signal sequence were identified. Clone has been deposited with the American Type Culture Collection and designated which contains the plasmid having the f.li~,.,,.. l. ~,ll.~t sequence as depicted 5 in Figure 13.

2174q43 ' ~
SEQUENCE LISTING
(1) GENER~L INFORMATION:
(i) APPLIChNT: Siegall, Clay B.
Gawlak, Susan L.
Marquardt, Hans (ii) TITLE OF INVENTION: A NEW RIBOSOME-INACTIVATING PROTEIN
ISOLATED FROM THE PLANT BRYONICA DIOICA
(iii) NUM3ER OF SEQUENCES: 15 (iv) UU~ UNL~;NCE ADDRESS:
(A ADDRESSEE: Bristol-Myers SquibB Company ( B STREET: 3 0 0 5 Fi rs t Avenue (C CITY: Seattle (D STATE: Washington (E COUNTRY: USA
(F ZIP: 98121 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compati}~le (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn R~lease #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PR~OR APPLICATION DATA:
(A) APPLICATION NUMBER: US C8/141, 891 (B1 FILING DATE: 25-OCT-1993 (viii) ATTORNEY~AGENT INFûRMATION:
(A) NAME: Poor, Brian W.
(B) REGISTRATION NUMBER: 32, 928 (C) REFERENCE/DOCKET NUMBER: ON0109A
(ix) TF.T.~.rn~MllNTCATION INFORMATION:
(A) TELEPHONE: 206-728-4800 (B) TELEFAX: 206-727-3601 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 32 amino ~Icids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FR~GMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonica dioica (F) TISSUE TYPE: Root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

2174 ~

.
Val Asp Ile Asn Phe Ser Leu Ile Gly Ala Thr Gly Ala Thr Tyr Lys Thr Phe Ile Arg Asn Leu Arg Thr Thr Leu Thr Val Gly Thr Pro Arg ~2) INFO~TION FOR SEQ ID NO:2:
(i) SEQUENCE CHMACTERISTICS:
(A) LENGTH: 16 amino acids ~B) TYPE: arlino acid ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide ~v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonica dioica (F) TISSUE TYPE: Root (xi) SEQUENCE DESCRII?TION: SEQ ID NO:2:
Leu Pro Tyr Gly Gly Asn Tyr Asp Gly Leu Glu Thr Ala Ala Gly Arg ~2) INFORMATION FOR SEQ ID NO:3:
(i ) SEQUENCE ChARACTERISTICS:
(A). LENGTH: l9 amino acids ~B) TYPE: arlino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 40 (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonica dioica (F) TISSUE TYPE: Root (xi) SEQUkNCE DESCRIPTION: SEQ ID NO:3:
Glu Asn Ile Glu Leu Gly Phe Ser Glu Ile Ser Ser Ala Ile Gly Asn 50 l S l0 15 Met Phe Arg 55 (2) INFORMl~TION FOR SEQ ID NO:4:
(i) SEQUENCE ChARACTERISTICS:
(A) LENGTH: 32 am~no acids (B) TYPE: amino acid (D) TOPOLOGY: line2r (ii) MOLECULE TYPE: peptide (v) FR~GMENT TYPE: internal 21 7 'q~3 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonica dioica (F) TISSUE TYPE: root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Phe Arg His Asn Pro Gly Thr Ser Val Pro Arg Ala Phe Ile Val Ile l S - l0 15 Ile Gln Thr Val Ser Glu Ala Ala Arg Phe Lys Tyr Ile Glu Gln Ar (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHAPACTERISTICS:
(A) LENGTH: 13 arino zcids ~B) TYPE: arlino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 25 (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonica dioica (F) TISSUE TYPE: root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:
Tyr Ile Glu Gln Arg Val Ser Glu Asn Val Gly Thr Lys 35 1 5 l0 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACT
(A) LENGTH: 39 arnino acld (B) TYPE: arlino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (vi) ORI6INAL SOURCE:
(A) ORGANISM: Bryonicz dioica (F) TISSUE TYPE: root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Lys Pro Asp Pro Ala Phe Leu Ser Leu Gln Asn Ala Trp Gly Ser 5 l0 15 Leu Ser Glu Gln Ile Gln Ile Ala Gln Thr Arg Gly Gly Glu Phe Ala Arg Pro Vzl Glu Leu Arg Thr (2) INFORMATION FOR SEQ ID NO:7:

2 1 749~
WO95111977 3 PCT/US91~12382 ~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: lS ami no acids (B) TYPE: ~mino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: intelnal (vi) ORIGINAL SOURCE:
~A) ORGANISM: Bryonica dioLca ~F) TISSUE TYPE: root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu Arg Thr Val Ser Asn Thr Pro Thr Phe Val Thr Asn Val Asn S l0 15 (2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE ChARACTERISTICS:
~A) LENGTH: 43 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lin~ar (iil MOLECULE TYPE: peptide 30 (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonia dioica (F) TISSUE TYPE: root (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asp Val Ser Phe Arg Leu Ser Gly Ala Thr Thr Thr Ser Tyr Gly Val l S l0 lS
Phe Ile Lys Asn Leu Arg Glu Ala Leu Pro Tyr Glu Arg Lys Val Tyr . 20 25 30 Asn Ile Pro Leu Leu Leu Arg His Xaa Ile Gly (2) INFORMATION FOR SEQ ID NO:9:
~i) SEQUENCE CHA~ACTERISTICS:
~A) LENGTH: 38 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i$) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Ricinus conununis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

WO 9S111977 L 1 7 ~ q 4 3 PCT/US94/12382 Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala S l0 15 Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu Thr Thr Gly Ala Asp Val 10 ~2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Momordia rnrhinrhin~ncis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Asp Val Ser Phe Arg Leu Ser Gly Ala A_p Pro Arg Ser Tyr Gly Met Phe Ile Lys Asp Leu Arg Asn Ala Leu Pro Phe Arg Glu Lys Val ~2) INFORMATION FOR SEQ ID NO:ll:
35 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 40 (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal ~vi) ORIGINAL SOURCE:
~A) ORGANISM: TrirhnsAn~h^~ kirilowii ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Asp Val Ser Phe Arg Leu Ser Gly-Ala Thr Ser Ser Ser Tyr Gly Val Phe Ile Ser Asn Leu Arg Lys Ala Leu Pro Asn Glu Arg Lys Leu ~2) INFORMATION FOR SEQ ID NO:12:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 31 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide WO 95/11977 2 1 7 ~ 9 4 3 PCT/US9~1112382 .
(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAl: SOURCE:
(A) ORGANISM: Luffa cyindrica (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asp Val Arg Phe Ser Leu Ser Gly Ser Ser Ser Thr Ser Tyr Ser Lys Phe Ile Gly Asp Leu Arg Lys Ala Leu Pro Ser Asn Gly Thr Val 15 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTh: 286 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 25 (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Momordica charantia 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Val Lys Cys Leu Leu Leu Ser Phe Leu Ile Ile Ala Ile Phe Ile Gly Val Pro Thr Ala Lys Gly Asp Val Asn Phe Asp .Leu Ser Thr Ala Thr Ala Lys Thr Tyr Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe Ser l~is Lys Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp Ser Arg Arg Phe Ile Leu Leu Asp Leu Thr Ser Tyr Ala Tyr 65 . 70 75 80 Glu Thr Ile Ser Val Ala Ile Asp Val Thr Asn Val Tyr Val Val Ala Tyr Arg Thr Arg Asp Val Ser Tyr Phe Phe Lys Glu Ser Pro Pro Glu Ala Tyr Asn Ile Leu Phe Lys Gly Thr Arg Lys Ile Thr Leu Pro Tyr Thr Gly Asn Tyr Glu Asn Leu Gln Thr Ala Ala E~is Lys Ile Arg Glu Asn Ile Asp Leu Gly Leu Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn Ala Gln Ser Ala Pro Ser Ala Leu Leu Tyr Leu Ile WO 9S/11977 ~J ,~ PCT/US94/12382 Gln Thr Thr Ala Glu Ala Ala Arg Phe Lys Tyr Il~ Glu Arg His Val Ala Lys Tyr Val Ala Thr Asn Phe Ly5 Pro Asn Leu Ala Ile Ile Ser 195 200: 205 Leu Glu Asn Gln Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln Gly Gly Lys Phe Arg Asn Pro Val Asp Leu Ile Lys Pro Thr Gly Glu Arg Phe Gln Val Thr Asn Val Asp Ser Asp Val Val Lys Gly Asn Ile Lys 3,eu Leu Leu Asn Ser Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile Thr Thr Met Thr Leu Leu Gly Glu Ser Val Val Asn (2) INFOR~ATION FOR SEQ ID NO:14:
(i) SEQUENCE CHMACTERISTICS:
(A) LENGTH: 962 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 30 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonia dioica ~F) TISSUE TYPE: leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

CCAAGGGTGT ACCATATACC TGTCCTGAGA AACGCAGCAG ~:CC~ L~, GCGCTTTCAA 240 GTGTACGTTG TTGCATATCG AGCTGGA~AC ACTGCTTACT TTCTCGCACA TGCATCAACA 360 55GAAGCCAACA Al~ TGCAGGCATC AATCATGTAA GACTTCC~TA TGCAGGGAAT 420 TCCCAAATAA GTAGTGCCAT TGGCAACATG TTCCGCCACA ACCCTGGTAC ~ 540 WO95111977 2 1 7 4 ~ 4 3 PCT/US9.1/12382 CAAAATGCTT GGGGCAGTCT CTCTGAACAA ATACAD~ATCG CACAAACTCG CGGAGGGGAA 720 llL~ f~_ CTGTCGAGCT TCGAACTGTT AGCAACACTC CGACTTTTGT GACCAATGTT 780 5AATTCGCCTG TTGTGA~AGG CATTGCACTT CTACTGTACT TTAGAGTTAA TGTTGGCACT 840 GATAATGTTT TCGCAATGTC CTTGTCAACC TACTAGTACT CATCAATCA~ ACTATACTGT 900 GTGCTTGTAT GTGCAAGTAT GGCAATAATA A~GACTTAAT CCTTTATGTT A~ AAA 6 (2) INFORMATION FOR SEQ ID NO:lS:
(i) SEQUENCE CHMACTERISTICS:
(A) LENGTII: 282 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bryonia dioica (F) TISSUE TYPE: leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
30 et Arg Ser Ile Gly Phe Tyr Ser Val Leu Ala Leu Tyr Val Gly Ala 5 10 lS
is Val Thr Glu Asp Val Asp Ile Asn Phe Ser Leu Ile Gly Ala Thr ly Ala Thr Tyr Lys Thr Phe Ile Arg Asn Leu Arg Thr Lys Leu Thr Val Gly Thr Pro Ary Val Tyr Asp Ile Pro Val 1eu Arg Asn Ala Ala Ala Gly Leu Ala Arg Phe Gln Leu Val Thr Leu Thr Asn T r Asn Gl Glu Ser Val Thr Val Ala Leu Asp Val Val Asn Val Tyr Val Val Ala Tyr Arg Ala Gly Asn Thr Ala Tyr Phe Leu Ala Asp Ala Ser Thr u 100 105 110 Gl Ala Asn Asn Val Leu Phe Ala Gly Ile Asn His Val Arg Leu Pro Tyr llS 120 125 ~5 Gly Gly Asn Tyr Asp Gly Leu Glu Thr Ala Ala Gly Arg Ile Ser Ar Glu Asn Ile Glu Leu Gly Phe Ser Glu Ile Ser Ser Ala Ile Gly Asn 145 150 lSS 160 Met Phe Arg His Asn Pro Gly Thr Ser Val Pro Arg Ala Phe Ile Val Il~ Ile Gln Thr Val Ser Glu Ala Ala Arg Phe Lys l~yr Ile Glu Gln WO 95/11977 , PCT/US94/12382 Arg Val Ser Glu Asn Val Gly Thr Lys Phe Lys Pro Asp Pro Ala Phe Leu Ser Leu Gln Asn Ala Trp Gly Ser Leu Ser Glu Gln Ile Gln Ile 210 215 ~ 220 Ala Gln Thr Arg Gly Gly Glu Phe Ala Arg Pro Val Glu Leu Arg Thr al Ser Asn Thr Pro Thr Phe Val Thr Asn Val Asn Ser Pro Val Val Lys Gly Ile Ala Leu Leu Leu Tyr Phe Arg Val Asn Val Gly Thr Asp Asn Val Phe Ala Met Ser Leu Ser Thr Tyr ~0 (2) INFORMATION FOR SEQ ID NO: 16:
~i) SEQUENCE ChARACTERISTICS:
(A) LENGTH: 17 base pairs ~5 (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE ChAPACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTIOIY: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

.... . _ _ . .. .. .. . _ WOg5/11977 2 ~ 74~43 PCTIUS94/12382 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: s~ngle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GACTCGAGTC GACATCGATT ~ 1 l l l l l l l l TTTTT 35 (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHAPACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l~near (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:20:

(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: s$ngle (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:

WO 95111977 , PCTNS94112382 1 49 (A) LENGTH: 28 base palrs ~B) TYPE: rlucleLc ~cld (C) STRANDEDNES5: single ~D) TOPOLOGY: linear ( ii ~ MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CGTTCACTAC ~TCTTAAGCC ACAGTGAC - 28 (2) INFORMATION FOR SEQ ID NO:24:
15 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
30 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nueleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:25:
GGCCACGCGT CGACTAGTAC G '~~ GGGNNG 36 (2) INFORMATION FOR SEQ ID NO:26:
45 (i) SEQUENCE CHA12ACTERISTICS:
. (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: lingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CAGGA~ACAG CTATGAC 17 (2) INFORMATION FOR SEQ ID NO:27:
60 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nueleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 95/11977 2 ~ 7 4 9 4 3 PCT/US9~/12382 (ii) I~OLECULE TY~E: cDNA
(xi) SEQUENCE DESCRIPTION: SE~2 ID NO:27:
1 C:. TTTTAC . 16 .~, ", ", ~

Claims (44)

C l a i m s

1. A ribosome-inactivating protein and functionally equivalent proteins comprising a single chain protein having a molecular weight of about 27,000 daltons by polyacrylamide gel electrophoresis under reducing and non-reducing conditions, wherein the amino terminal amino acid residue sequence comprises the following contiguous amino acid sequence:
Val Asp Ile Asn Phe Ser Leu Ile Gly Ala Thr Gly Ala Thr Tyr Lys Thr Phe Ile Arg Asn Leu Arg Thr Thr Leu Thr Val Gly Thr Pro Arg (Seq. ID #1).

2. The ribosome-inactivating protein of claim l having an EC50 of about 0.017 mM in a rabbit reticulocyte lysate system and an LD50 in mice of greater than 10 mg/kg when administered intravenously and about 8 mg/kg when administered intraperitoneally.

3. The ribosome-inactivating protein of claim 1 or 2, wherein the protein is isolated from the root of Bryonia dioica.

4. The ribosome-inactivating protein of claim 1, wherein the protein comprises a contiguous internal amino acid residue sequence of:
(a) Leu Pro Tyr Gly Gly Asn Tyr Asp Gly Leu Glu Thr Ala Ala Gly Arg (Seq. ID #2);

(b) Glu Asn Ile Glu Leu Gly Phe Ser Glu Ile Ser Ser Ala Ile Gly Asn Met Phe Arg (Seq. ID #3);
(c) Phe Arg His Asn Pro Gly Thr Ser Val Pro Arg Ala Phe Ile Val Ile Ile Gln Thr Val Ser Glu Ala Ala Arg Phe Lys Tyr Ile Glu Gln Arg (Seq. ID#4);
(d) Tyr Ile Glu Gln Arg Val Ser Glu Asn Val Gly Thr Lys (Seq. ID #S);
(e) Phe Lys Pro Asp Pro Ala Phe Leu Ser Leu Gln Asn Ala Trp Gly Ser Leu Ser Glu Gln Ile Gln Ile Ala Gln Thr Arg Gly Gly Glu Phe Ala Arg Pro Val Glu Leu Arg Thr (Seq.ID #6); or (f) Leu Arg Thr Val Ser Asn Thr Pro Thr Phe Val Thr Asn Val Asn (Seq. ID #7).

5. A composition comprising the ribosome-inactivating protein of claim 1 linked to a ligand to form a toxin-ligand conjugate.

6. The composition of claim 5, wherein the ligand comprises an immunoglobulin, adhesion molecule, or a polypeptide, peptide or non-peptide ligand.

7. The composition of claim 6, wherein the ligand is selected from the group consisting of transferrin, an epidermal growth factor, bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, interleukin-2, interleukin-6, transforming growth factors, steroid, carbohydrate and lectin.

8. The composition of claim 6, wherein the ligand is an immunoglobulin

9. The composition of claim 8, wherein the immunoglobulin is an antigen recognizing fragment, a chimeric antibody, a bifunctional antibody or a hybrid antibody.

10. The composition of claim 9, wherein the antigen-recognizing fragment is a Fab',(Fab')2, Fv or Fab fragment.

11. The composition of claim 9, wherein the immunoglobulin is immunospecific for a Lewis Y-related antigen and is internalized by carcinoma cells.

12. The composition of claim 9, wherein the chimeric immunoglobulin is chimeric BR96 as produced by the hybridoma deposited with the American Type Culture Collection and designated ATCC HB 10460.

13. A pharmaceutical comprising the ribosome-activating protein of claim 1 and a pharmaceutically acceptable carrier or adjuvant.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable carrier or adjuvant is human serum albumin, albumin, an ion exchanger, alumina, lecithin, a buffer substance, salt or electrolyte.

15. A pharmaceutical composition comprising an immunotoxin comprising bryodin 2 and a ligand, and a pharmaceutically acceptable carrier or adjuvant.

16. The pharmaceutical composition of claim 15, wherein the ligand is an immunoglobulin.

17. The pharmaceutical composition of claim 16, wherein the immunoglobulin is an antigen recognizing fragment, a chimeric antibody, a bifunctional antibody or a hybrid antibody.

18. The composition of claim 17, wherein the immunoglobulin is a chimeric antibody.

19. The composition of claim 18, wherein the chimeric antibody is chimeric BR96 as produced by the hybridoma deposited with the American Type Culture Collection and designated ATCC HB10460.

20. An isolated oligonucleotide sequence encoding a ribosome-activating protein from Bryomia dioica the protein comprising the amino acid sequence of Sequence ID #15, or a complement of the isolated olignucleotide sequence.

21. The isolated oligonucleotide sequence of claim 20 comprising the nucleotide sequence of Sequence ID #14 from about nucleotide number 28 to about nucleotide number 873.

22. The isolated oligonucleotide sequence of claim 20 comprising the nucleotide sequence of Sequence ID #14 from about nucleotide number 91 to about nucleotide number 873.

23. The isolated nucleotide sequence of claim 20, wherein the nucleotide sequence encodes a biologically active fragment of bryodin 2 which inhibits protein synthesis in vitro.

24. A recombinant vector comprising an oligonucleotide sequence encoding a ribosome-inactivating protein from Bryodin dioica, the protein comprising the amino acid sequence of Sequence ID #15.

25. The recombinant vector of claim 24, further comprising transcriptional and translational control sequences operably linked to the oligonucleotide sequence encoding the ribosome-inactivating protein.

26. The recombinant vector of claim 24 wherein the vector is pSE20.0 as deposited with the American Type Culture Collection and designated ATCC

27. A host cell transfected with a recombinant vector of claim 24.

28. A host cell transfected with a recombinant vector of claim 26

29. A method for the recombinant expression of bryodin 2 comprising transfecting a host cell with an expression vector comprising an oligonucleotide sequence encoding the contiguous amino acid sequence of Sequence ID #15, growing the transfected host cells, inducing the transfected host cells to express recombinant bryodin 2 and isolating the expressed recombinant bryodin 2.

30. The method of claim 29, wherein the host cell is a bacteria, a plant cell, ayeast or a mammalian cell.

31. A method for producing a, recombinant bryodin 2-ligand fusion protein comprising a transfected host cell with an expression vector comprising an oligonucleotide sequence encoding the contiguous amino acid sequence of Sequence ID) #2 from about amino acid residue 22 to about amino acid residue 282 operatively linked with anoligonucleotide sequence which encodes a ligand, growing the transfected host cells, inducing the transfected host cells to express the recombinant bryodin 2-ligand fusion protein, and isolating the expressed recombinant fusion protein.

32. The method of claim 31, wherein the host cell is a bacteria, a plant, a yeast or a mammalian cell.

33. The method of claim 32, wherein the ligand is a large molecular weight protein, a small molecular weight protein, a polypeptide, or a peptide-ligand.

34. The method of claim 33, wherein the ligand is an immunoreactive ligand.

35. The method of claim 34, wherein the immumoreactive ligand is an antigen recognizing immunoglobulin, or an antigen-recognizing fragment thereof, a chimeric antibody, a bifunctional antibody, a hybrid antibody or a single chain antibody.36. The method of claim 35, wherein the antigen recognizing fragment is a

Fab',F(ab')2, Fv or Fab fragment of an immunoglobulin.

37. A method for killing a target cell comprising contacting the target cell with an effective amount of a toxin-ligand conjugate of claim 5, wherein the ligand specifically binds to or reactively associates with a receptor moiety on the surface of the target cell, for a time sufficient to kill the target cell.

38. The method of claim 37, wherein the ligand comprises an immunoglobulin, adhesion molecule, or a polypeptide, peptide or nonpeptidyl ligand.

39. The method of claim 37, wherein the wherein the immunoglobulin is an antigen binding fragment, a chimeric antibody, a bifunctional antibody or a hybrid antibody.

40. The method of claim 39, wherein the chimeric immunoglobulin is chimeric BR96 as produced by the hybridoma deposited with the American Type Culture Collection and designated ATCCHB 10460.

41. A method for inhibiting the proliferation of mammalian tumor cells comprising contacting the mammalian tumor cells with a proliferation inhibiting amount of a tumor targeted toxin joined to a ligand specific for a tumor-associated antigen so as to inhibit proliferation of the mammalian tumor cells.

42. The method of claim 41, wherein the ligand comprises an immunoglobulin, adhesion molecule, or a polypeptide, peptide or nonpeptidyl ligand.

43. The method of claim 41, wherein the immumnoglobulin is an antigen binding fragment, a chimeric antibody, a bifunctional antibody or a hybrid antibody.

44. The method of claim 43, wherein the chimeric immunoglobulin is chimeric BR96 as produced by the hybridoma deposited with the American Type Culture Collection and designated ATCC HB10460.