WO2002058624A9 - Generation in situ specifique d'un site de l'allicine au moyen d'un systeme de distribution cible d'alliinase pour le traitement des cancers, tumeurs, maladies infectieuses et autres maladies sensibles a l'allicine - Google Patents

Generation in situ specifique d'un site de l'allicine au moyen d'un systeme de distribution cible d'alliinase pour le traitement des cancers, tumeurs, maladies infectieuses et autres maladies sensibles a l'allicine

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Publication number
WO2002058624A9
WO2002058624A9 PCT/US2001/049384 US0149384W WO02058624A9 WO 2002058624 A9 WO2002058624 A9 WO 2002058624A9 US 0149384 W US0149384 W US 0149384W WO 02058624 A9 WO02058624 A9 WO 02058624A9
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WO
WIPO (PCT)
Prior art keywords
alliinase
conjugate
cells
allicin
alliin
Prior art date
Application number
PCT/US2001/049384
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English (en)
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WO2002058624A2 (fr
WO2002058624A3 (fr
Inventor
Aharon Rabinkov
Talia Miron
David Mirelman
Meir Wilchek
Original Assignee
Yeda Res & Dev
Aharon Rabinkov
Talia Miron
David Mirelman
Meir Wilchek
Mcinnis Patricia
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Filing date
Publication date
Application filed by Yeda Res & Dev, Aharon Rabinkov, Talia Miron, David Mirelman, Meir Wilchek, Mcinnis Patricia filed Critical Yeda Res & Dev
Priority to AU2002246727A priority Critical patent/AU2002246727A1/en
Priority to US10/451,849 priority patent/US7445802B2/en
Priority to IL15661701A priority patent/IL156617A0/xx
Publication of WO2002058624A2 publication Critical patent/WO2002058624A2/fr
Publication of WO2002058624A3 publication Critical patent/WO2002058624A3/fr
Priority to IL156617A priority patent/IL156617A/en
Publication of WO2002058624A9 publication Critical patent/WO2002058624A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to conjugates of alliinase with a protein carrier that targets the alliinase to specific cells.
  • the present invention further relates to a method for producing allicin in si tu by administering this conjugate followed by administration of alliin.
  • Allicin is a chemically unstable colorless liquid that it believed to be responsible for both the odor and much of the biological activity ascribed to garlic. Thus, allicin possesses a remarkably broad spectrum of antibiotic activities, including antibacterial activity against a wide range of Gram- negative and Gram-positive aerobic and anaerobic bacteria, as well as antifungal, antiprotozoal, antiviral, antiparasitic, and insecticidal activities. Miron et al (international admiron WO 97/39115) discloses a biotechnological process for preparing pure allicin in practically unlimited amounts .
  • Allicin is a very chemically active molecule, which readily reacts with compounds in the body and disappears within a few minutes after being mixed with blood.
  • In vivo activities of allicin can be defined as unique activities, that is, those exhibited only by allicin itself, and non-unique activities, those which are exhibited by allicin derivatives or secondary products produced from allicin during treatment with allicin.
  • the most valuable intrinsic activity of allicin is its prominent and broad spectrum antibiotic activity. However, this activity has been noted only for allicin and one of its derivatives, ajoene.
  • allicin against several microorganisms is very important, especially in cases in which effective therapy has still not yet been developed.
  • the cytotoxic properties of allicin are significantly higher than those of other sulfur compounds derived from garlic, and the present inventors have demonstrated that purified allicin was effective against cancer cells in concentrations significantly lower than those effective to kill normal cells (Hirsh et al, 2000) .
  • the present invention overcomes the problem of in vivo inactivation of allicin by generating allicin in vivo at the site at which it should exert it desired biological activity.
  • the enzyme alliinase which catalyzes the synthesis of allicin from its precursor alliin, is coupled to a carrier protein which directs the alliinase to a target.
  • the stable conjugate retains both the alliinase enzymatic activity and the epitope binding activity of the carrier protein to antigens or receptors on the surface of cells of interest without damaging the cells.
  • a patient is treated with this conjugate followed by administration of the precursor alliin.
  • the alliin is converted by the alliinase to allicin, leading to inhibition of cell growth at that site.
  • the targeting carrier is a cell- or tissue-specific monoclonal antibody that recognizes specific receptors on a cell surface, or a derivative of the monoclonal antibody such as a F(ab) 2 dimer, a F(ab) monomer, Fv and single chain natural or recombinant Fv.
  • the monoclonal antibody recognizes a specific antigen on the surface of cancer cells.
  • this monoclonal antibody recognizes the ErbB-2 receptor on the surface of cancer cells.
  • One example of such a monoclonal antibody is the antibody herein designated N28 or a F(ab) 2 dimer or F(ab) monomer thereof.
  • the present invention relates to a method for treating a disorder or disease treatable with allicin that comprises administering to an individual in need thereof a conjugate of the enzyme alliinase with a carrier protein that targets the conjugate to a desired tissue or organ in the body, followed by administering alliin, whereby allicin is generated at the tissue or organ site, where it can exert its biological activity.
  • Figure 1 shows inhibition of CB-2 cell proliferation by different concentrations of allicin.
  • Figures 2A-2C show tissue cultured CB-2 cells ( Figure 2A) , N87 cells ( Figure 2B) , and normal human foreskin fibroblasts (Figure 2C) stained with Trypan blue, untreated (upper left pictures) or after exposure to alliin (upper right pictures) or to allicin (lower pictures) x 400.
  • Figure 3 shows determination of allicin produced after binding of mAB ErbB-2-alliinase conjugates or F(ab)- alliinase to CB-2 (black columns) and N87 (gray columns) cancer cells which express ErbB-2 receptors.
  • Figure 4 shows determination of allicin produced as a function of different amounts of conjugates bound to CB-2 cells: mAb ErbB-2 (black columns); mAb ErbB-2 alliinase (light gray columns) ; mAb ErbB-2-alliinasePEG5000 (dark gray columns) .
  • Figures 5A-5B show inhibition of [ 3 H] -thymidine incorporation into CB-2 cells upon treatment with conjugate of F (ab) ErbB-2-alliinase alone ( Figure 5A) or followed by the addition of alliin ( Figure 5B) . Each treatment was conducted in triplicate.
  • Figure 6 shows inhibition of [ 3 H] -thymidine incorporation in 32D (blank columns) and N87 (gray columns) cells upon treatment with: none, mAbErbB-2-alone, or the conjugate mAbErbB-2-alliinase, in the absence (-) or presence (+ ) of added alliin. 3 H thymidine incorporated into the DNA was determined. The experiments were conducted in triplicate.
  • Figure 7 shows the effect of mAb ErbB-2-alliinase conjugate on the viability of cultured CB-2 (upper pictures) and N87 (lower pictures) cancer cells in the presence or absence of alliin (x 400).
  • Figure 8 depicts the concept of using mAb-alliinase conjugate together with alliin for in si tu production of allicin in anticancer therapy.
  • Figure 9A shows the distribution of 125 I-anti ErbB2 monoclonal antibodies in organs of mice pretreated with N-87 tumor cells. Treated mice were sacrificed after 8, 24 and 72 hours .
  • Figure 9B shows the accumulation of 125 I mAb-alliinase conjugate in the tumors at 8, 16, 24, 48, 72 and 120 hours.
  • Figure 10 shows distribution of 125 I-anti ErbB2- alliinase conjugate in organs of mice pretreated with N-87 cells. Treated mice were sacrificed after 8, 24, and 48 hours.
  • Figure 11 shows the effect of conjugated alliinase- mAb N-28 ⁇ alliin on tumor growth in mice pre-treated with N-87 cancer cells. Mice treated with mAb-Alliinase conjugates without administration of alliin D; with administration of alliin H.
  • a new approach for treating cancer and bacterial infections whereby alliinase is conjugated to a carrier protein for specific delivery and targeting of the alliinase to the surface of target cells.
  • the cells are then exposed to alliin, at which time the alliinase converts the alliin in si tu to allicin, which kills the target cells.
  • This concept is illustrated in Figure 8, and the results shown herein validate the efficacy of the invention.
  • the present invention provides a method to generate allicin in si tu at a desired location in the body, wherein allicin is generated directly at the site to be treated so that the allicin does not deteriorate prior to reaching the site to be treated.
  • the allicin-producing enzyme, alliinase is delivered directly to the sites where allicin is desired to produce its effect. Following administration of the biologically inactive and non-toxic alliinase substrate alliin, allicin production occurs only at the place where the alliinase is located.
  • allicin has the advantages of this approach based on- the specific features of allicin, namely, its potent biological activity, its ability to rapidly penetrate through biological membranes, its extremely short lifetime in the body as well as its low toxicity, and its conversion into non-toxic and even beneficial secondary products.
  • the biologically potent active molecule allicin is generated by the alliin-alliinase system at specific targeted sites, and thus allicin will exert its toxic effect locally.
  • the present invention is a new concept in targeting therapy.
  • the present invention uses the site-directed in si tu production of allicin to combat infectious disease, cancer, and other allicin-sensitive disorders.
  • the present invention provides the enzyme alliinase in an enzymatically active form conjugated with a targeting carrier which guides the enzyme to the cell or to the microorganism of interest in the body.
  • Any alliinase or enzyme which has alliinase activity may be used in the present invention, whether the entire molecule or a derivative or fragment thereof, as long as it retains its catalytic activity and ability to generate allicin or analogs thereof in a lyase reaction from alliin substrates or analogs thereof. While natural alliinase from garlic is the preferred enzyme for use in the present invention, alliinase from any source, including onion, Brassicaceae , Fabaceae, broccoli, and even bacteria can be used.
  • Representative amino acid sequences for alliin from a variety of plant sources include but are not limited to those of GenBank accession numbers S35460, S29302, S29300, S29301, BAB68045, BAB68042, AAK95663, AAK96552, AAK95661, AAK95660, AA 95659, AAK95698, AAK95657, AAK95656, NP177213, NP173746, P31757, AAG52476, AAG52348, AAG12844, AAG00599, AAF81248, AAF36437, Q01594, P31756, AAD51706, AAD51705, AAD51704, AAD51703, AAD51702, AAD51701, AAD43130, AAD32696, AAD26853, AAD21617, BAA20358, AAB32477, CAA78268, CAA78267, CAA78266, CAA63482, AAA92463, and AAA32639.
  • An analog of alliinase has an amino acid sequence essentially corresponding to any one of the amino acid sequences of alliinase available in GenBank disclosed above.
  • the term "essentially corresponding to” is intended to comprehend analogs with minor changes to the sequence of the protein or polypeptide which do not affect the basic characteristics thereof, particularly insofar as its catalytic activity and ability to generate allicin or analogs thereof in a lyase reaction from alliin substrates or analogs thereof is concerned.
  • the type of changes which are generally considered to fall within the "essentially corresponding to” language are those which would result from conventional mutagenesis techniques of the DNA encoding alliinase, resulting in a few minor modifications, and screening for the desired activity in the manner discussed above.
  • the analog is a variant of a native sequence or a biologically active fragment thereof which has an amino acid sequence having at least 70% identity to a native amino acid sequence and retains the biological activity thereof. More preferably, such a sequence has at least 85% identity, at least 90% identity, or most preferably at least 95% identity to a native sequence.
  • Analogs in accordance with the present invention may also be determined in accordance with the following procedure.
  • Polypeptides encoded by any nucleic acid, such as DNA or RNA, which hybridize to the complement of the native DNA or RNA under highly stringent or moderately stringent conditions, as long as that polypeptide maintains the alliinase catalytic activity of a known native sequence are also considered to be within the scope of the present invention.
  • nucleotide sequence of variants of known native alliinase in question may be determined by hybridization of a cDNA library using a probe which is based on the identified polynucleotide, under highly stringent conditions. Stringency conditions are a function of the temperature used in the hybridization experiment and washes, the molarity of the monovalent cations in the hybridization solution and in the wash solution (s) and the percentage of formamide in the hybridization solution.
  • sensitivity by hybridization with a probe is affected by the amount and specific activity of the probe, the amount of the target nucleic acid, the detectability of the label, the rate of hybridization, and the duration of the hybridization.
  • the hybridization rate is maximized at a Ti (incubation temperature) of 20-25°C below Tm for DNA: DNA hybrids and 10- 15 °C below Tm for DNA: RNA hybrids. It is also maximized by an ionic strength of about 1.5M Na + .
  • the rate is directly proportional to duplex length and inversely proportional to the degree of mismatching.
  • Hybrid stability is a function of duplex length, base composition, ionic strength, mismatching, and destabilizing agents (if any) .
  • Tm 79.8°C + 18.5 (log M) + 0.58 (%GC) - 11.8 (%GC) 2 - 0.56(% form) - 820/L
  • Tm is reduced by 0.5-1.5°C (an average of 1°C can be used for ease of calculation) for each 1% mismatching.
  • the Tm may also be determined experimentally. As increasing length of the hybrid (L) in the above equations increases the Tm and enhances stability, a full-length native alliinase DNA sequence can be used as the probe.
  • Filter hybridization is typically carried out at 68°C (lower temperatures are used if formamide is added to compensate for the lowering of the hybridization temperature) , and at high ionic strength (e.g., 5-6 X SSC), which is non- stringent, and followed by one or more washes of increasing stringency, the last one being of the ultimately desired stringency.
  • high ionic strength e.g., 5-6 X SSC
  • the equations for Tm can be used to estimate the appropriate Ti for the final wash, or the Tm of the perfect duplex can be determined experimentally and Ti then adjusted accordingly.
  • Hybridization conditions should be chosen so as to permit allelic variations and splice variants, but avoid hybridizing to other non-alliinase genes.
  • highly stringent conditions are considered to be a Ti of 5°C below the Tm of a perfect duplex, and a 1% divergence corresponds to a 0.5-1.5°C reduction in Tm.
  • Use of a Ti of 5-15°C below, more preferably 5-10°C below, the Tm of the double stranded form of the probe is recommended for probing a cDNA library.
  • highly stringent conditions are those which are tolerant of up to about 15% sequence divergence.
  • examples of highly stringent (5-15°C below the calculated Tm of the hybrid) conditions use a wash solution of 0.1 X SSC (standard saline citrate) and 0.5% SDS at the appropriate Ti below the calculated Tm of the hybrid.
  • the ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids.
  • a common hybridization condition that can be used with the highly stringent wash conditions described above is hybridization in a solution of 6 X SSC (or 6 X SSPE), 5 X Denhardt ' s reagent, 0.5% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA at an appropriate incubation temperature Ti.
  • “Functional derivatives” as used herein covers chemical derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the catalytic activity of the corresponding alliinase enzyme as described herein.
  • Derivatives may have chemical moieties, such as carbohydrate or phosphate residues, provided such a fraction has the same catalytic activity and remains pharmaceutically acceptable.
  • Suitable derivatives may include aliphatic esters of the carboxyl of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed with acyl moieties.
  • Such derivatives may also include for example, polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the complex or the portions thereof in body fluids.
  • Non-limiting examples of such derivatives are described below.
  • Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha- bro o-beta- (5-imidazoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2- pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole .
  • Histidyl residues are derivatized by reaction with diethylprocarbonate - at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Parabromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the ⁇ effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- methylisourea; 2, 4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1, 2-cyclodexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R'-N-C-N- R') such as l-cyclohexyl-3- [2-morpholinyl- (4- ethyl) ] carbodiimide or l-ethyl-3- (4-azonia-4, 4-dimethlypentyl) carbodiimide.
  • carbodiimides R'-N-C-N- R'
  • carbodiimides R'-N-C-N- R'
  • carbodiimides Rosinyl or glutamyl
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions .
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope
  • derivatives is intended to include only those derivatives that do not change one amino acid to another of the twenty commonly-occurring natural amino acids.
  • salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the complex of the invention or analogs thereof.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid.
  • mineral acids such as, for example, hydrochloric acid or sulfuric acid
  • organic acids such as, for example, acetic acid or oxalic acid.
  • any such salts must have substantially similar biological activity to the complex of the invention or its analogs.
  • fragment of the enzyme alliinase or a variant thereof is intended to cover any fragment of alliinase or an analog thereof that retains the catalytic activity and ability to generate allicin in a lyase reaction from alliin substrates.
  • fragments can be readily generated from alliinase where successive residues can be removed from either or both the N-terminus or C-terminus of alliinase, or from peptides obtained thereof by enzymatic or chemical cleavage of the polypeptide.
  • multiple substitutions are not involved in screening for catalytically active fragments of alliinase.
  • the targeting carrier in one preferred embodiment, is a monoclonal antibody of either human or animal origin. It may be a natural, recombinant or humanized antibody, or it may be a derivative thereof such as a F(ab) 2 dimer or F(ab) monomer, Fv or a natural or recombinant single-chain Fv.
  • the antibody must be one that recognizes a specific receptor on the cell surface to be targeted and are not internalized.
  • Derivatives of monoclonal antibodies are those molecules that recognize the same receptor on the cell surface as the monoclonal antibody and thus can be used interchangeably with the intact monoclonal antibody in the present invention.
  • antibodies are used with respect to the antibody embodiments of the present invention, this is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs) , as well as proteolytic fragments thereof such as the Fab or F(ab')2 fragments.
  • the DNA encoding the variable region of the antibody can be inserted into other antibodies to produce chimeric antibodies (see, for example, Cabilly et al, U.S. patent no. 4,816,567) or into T- cell receptors to produce T-cells with the same broad specificity (see Eshhar et al, 1990 and Gross et al, 1989) .
  • Single-chain antibodies can also be produced and used.
  • Single- chain antibodies can be single-chain composite polypeptides having antigen binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked VH- VL or single-chain FV) .
  • Both VH and VL may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in Huston et al, U.S. patent no. 5,091,513 (the entire content of which is hereby incorporated herein by reference) .
  • the separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker.
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler et al, (1975); David et al, U.S. patent no. 4,376,110; Harlow et al, (1988); and Colligan et al, (2001) , the entire contents of which references are incorporated entirely herein by reference.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof.
  • the hybridoma producing the mAbs of this invention may be cultivated in vi tro or in vivo.
  • High titers of mAbs can be obtained by in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristane-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs.
  • JMAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Chimeric antibodies are primarily used to reduce immunogenicity during application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric or humanized mAbs are used.
  • Chimeric and humanized antibodies and methods for their production are well-known in the art, such as Cabilly et al (1984); Morrison et al (1984); Boulianne et al (1984); Cabilly et al, European Patent 0 125 023 (1984);
  • a "molecule which includes the antigen-binding portion of an antibody is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, or generated in vi tro, such as by phage display technology for constructing recombinant antibodies, but also the antigen-binding reactive fraction thereof (also referred to herein as a derivative thereof) , including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab') 2 fragment, the variable portion of the heavy and/or light chains thereof, and chimeric or single-chain antibodies incorporating such reactive fraction, or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive fraction.
  • Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.
  • the targeting carrier is a lectin, a carbohydrate-binding protein, a hormone, or a ligand which has specific receptor binding properties for specific cells, or a special polymer with tropism to particular tissues and cells.
  • Examples of monoclonal antibodies that can be coupled to alliinase for use in the present invention include, without being limited to, human or humanized monoclonal antibodies, some of which are commercially available, against a variety of specific antigens on the surfaces of cancer cells and metastatic cells.
  • Examples of such anticancer monoclonal antibodies are the monoclonal antibodies against the ErbB-2 receptor present on the surface of many cancer cells, particularly the antibodies described in Bacus, U.S. patent no. 5,514,554; and Sela et al, European patent no. EP 0 554 441, which are herein incorporated by reference in their entirety.
  • Of particular interest is the monoclonal antibody against ErbB- 2 receptor herein designated N28, which was deposited at the CNCM, Institut Pasteur, Paris, France, August 1992, under the Accession Number 1-1261.
  • antibodies that can be coupled to alliinase for cancer therapy according to the present invention include monoclonal antibodies against a variety of cancer cells and metastatic antigens related to, for example, breast, colorectal, prostate, or bladder cancers, and of different leukemias such as the antibodies against the CD-20 receptor containing B-lymphocytic or lymphoblastic leukemia cells.
  • monoclonal antibodies against a variety of cancer cells and metastatic antigens related to, for example, breast, colorectal, prostate, or bladder cancers and of different leukemias such as the antibodies against the CD-20 receptor containing B-lymphocytic or lymphoblastic leukemia cells.
  • Examples of particular cancer-specific antibodies which may be used in the present invention are listed in the "e-book" Monoclonal Antibody Index, Vol 1: Cancer (2000) .
  • the targeting carrier may be a monoclonal antibody against an infectious disease.
  • viruses e.g., hepatitis B and C, HIV, CMV, etc.
  • infectious agents such as viruses, e.g., hepatitis B and C, HIV, CMV, etc.
  • infectious agents such as viruses, e.g., hepatitis B and C, HIV, CMV, etc.
  • other infectious diseases such as tuberculosis, lung inflammation, (bacterial, viral or fungal) , bronchitis (viral or bacterial) , leprosy, meningitis (viral, bacterial, or fungal) , plague, typhus or paratyphus A and B, influenza, herpes zoster, cholera, malaria, measles, acute and chronic liver infections (including from hepatitis B and C) , rabies, and AIDS.
  • Some such examples are monoclonal antibodies against hepatitis viruses or other viruses such as HIV, against fungi such as Candida albicans, against bacterial infections such as
  • Staphylococci or Streptococci causing bateriemia against parasites which appear in the circulation such as Trypanosom.es, Plasmodi um, Leishmania, and the like.
  • a monoclonal antibody specific for a given pathogen or disease can be selected from among those listed in the "e-book" Monoclonal Antibody Index, Vol. 2: Transplant, Infection and Heart, (2001) .
  • Alliinase can be attached to a targeting carrier by chemical means by generating covalently bound conjugates, as well as by physical means such as by affinity binding, entrapping the enzyme within a polymer matrix or membrane (liposome) , or microencapsulating the enzyme within semipermeable polymer membranes.
  • the alliinase can be bound to the carrier directly or through a spacer.
  • the carriers used in the present invention are antibodies or other ligands which recognize cell receptors, as well as specific polymers, liposomes, etc. Coupling alliinase to these carriers involves mild reactions between amino acid residues of the enzyme and functional groups of the carrier.
  • Functional groups of carriers that can be used in the present invention are thiol, hydroxyl, amino, and carboxyl groups, which must be activated for coupling with alliinase.
  • Coupling can be effected by various chemical crosslinking methods, forming peptide (amide) , azo, thioether, disulfide, and other bonds. 7 ⁇ ny of these methods can be used for this invention.
  • carboxyl groups of the carrier are converted to reactive derivatives such as N- hydroxysuccinimide ester. And these derivatives form peptide bonds with free amino groups of alliinase. It is also possible to form peptide bonds between free carboxyl or amino groups of the enzyme, and amino groups or carboxyl groups of the carrier, respectively, using condensing agents such as cabodiimides and Woodward's reagent K.
  • Another possibility is to use a molecule which serves as a spacer, e.g., epsilon-aminocaproic acid or 3- (2- pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) .
  • the spacer is first attached either to the carrier or to alliinase through an amino group. Then, using the methods described above, the two entities are combined.
  • alliinase can be coupled with various biocompatible synthetic polymers such as ethoxypolyethylene glycol ( PEG) prior to being coupled to the carrier molecule.
  • PEG ethoxypolyethylene glycol
  • Pegylation of alliinase is carried out by standard techniques, well known to one skilled in the art.
  • Alliinase can be coupled with the targeting carrier using, for example, biotinlyated alliinase or haptenized alliinase that can interact with avidin on the antibody.
  • Another approach for preparing conjugates of the present invention consists in developing recombinant carrier- alliinase fusion proteins, for example, mAb-alliinase, consisting of single molecular entities. Genetically engineered fusion proteins may be constructed by cloning the gene sequences of antibody light chains and heavy chains fused to sequences encoding alliinase. As an example, mRNA from hybridoma cells expressing a monoclonal antibody is isolated.
  • cDNA is reverse transcribed and amplified by polymerase chain reaction.
  • Specific regions encoding heavy and light chains of an immunoglobulin e.g., variable and/or constant regions, can be amplified by the selection of appropriate oligonucleotide primers targeting the desired region (s).
  • the cDNA is sequenced, mapped by restriction endonucleases, and cloned into an appropriate transfer vector.
  • the immunoglobulin sequences encoding an antigen binding domain i.e., the variable light chain and variable heavy chain regions, are contained in the transfer vector.
  • a truncated or full-length portion of the constant region encoding the original or another immunoglobin can be joined in frame with the variable region, to allow expression of the joined regions.
  • a preferred embodiment of the invention encodes a chimeric mAb, comprised of murine variable regions linked to their corresponding human constant regions of the heavy and light chains.
  • An appropriate DNA sequence, encoding at least one alliinase peptide, is then ligated proximate to a region of an immunoglobulin gene encoding the carboxy-terminus, preferably a constant region, most preferably the constant region of a heavy chain.
  • the best site for attachment for each alliinase may be different and may be easily determined via experimental methods. For example, none or various lengths of amino acid encoding linkers may be inserted between the alliinase and the carboxy-terminus of the immunoglobulin gene. The resulting expression products can then be tested for biologic activity.
  • the completed engineered gene for the fusion protein is inserted into an expression vector, which can be introduced into eukaryotic or prokaryotic cells by gene transfection methods, e.g., electroporation or the calcium phosphate method.
  • the fusion protein product can then be expressed in large-scale cell culture and purified.
  • the C-terminal end of alliinase, or any biologically active analog or fragment thereof may be fused to the N- terminal end of an immunoglobulin chain, preferably a single- chain antibody (scFv) or an antigen-binding fragment thereof.
  • the reverse constructs can also be prepared, where the C- terminal end of the antibody chain is fused to the N-terminal end of the alliinase molecule.
  • a flexible peptide linker for example Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO:l) repeats, may be employed.
  • these linkers are up to about 30 amino acids in length.
  • the present invention also concerns DNA sequences encoding the above fusion protein of the invention, as well as DNA vectors carrying such DNA sequences for expression in suitable prokaryotic or eukaryotic host cells.
  • suitable prokaryotic or eukaryotic host cells e.g., bacterial
  • eukaryotes e.g., yeast
  • eukaryotic species e.g., insect and mammalian cells.
  • the present invention provides a pharmaceutical composition comprising a conjugate of the invention and a pharmaceutically acceptable carrier. While any suitable formulation of the composition is encompassed by the invention, preferably, it will be adapted for intravenous administration.
  • Pharmaceutical compositions according to the present invention can be administered by any convenient route, including parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, or transdermal . Alternatively or concomitantly, administration may be by the oral route. The dosage administered depends upon the age, health, and weight of the recipient, nature of concurrent treatment, if any, and the nature of the effect desired.
  • Compositions within the scope of the present invention include all compositions wherein the conjugate is contained in an amount effective to achieve its intended purpose.
  • Typical preferred dosages comprise 0.01 to 100 mg/kg body weight.
  • the preferred dosages comprising 0.1 to 100 mg/kg body weight.
  • the most preferred dosages comprise 1 to 50 mg/kg body weight.
  • compositions for administering the active ingredients of the present invention preferably contain, in addition to the pharmacologically active compound, suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations contain from about 0.01 to about 99 percent by weight, preferably from about 20 to 75 percent by weight, active compound(s), together with the excipient.
  • active compound(s) for purposes of the present invention, all percentages are by weight unless otherwise indicated.
  • the compounds of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes.
  • the pharmaceutically acceptable carriers include vehicles, adjuvants, excipients, or diluents that are well known to those skilled in the art and which are readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and which has no detrimental side effects or toxicity under the conditions of use.
  • compositions of the present invention are determined partly by the particular active ingredient, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention. While the preferred route for administering the conjugates of the present invention is oral, formulations can also be prepared for aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intratracheal, rectal, and vaginal administration.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, such as water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides .
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the active ingredient may be present both in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipid layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetyl phosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • phospholipids such as lecithin and sphingomyelin
  • steroids such as cholesterol
  • more or less ionic surface active substances such as dicetyl phosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • Liquid formulations may include diluents such as water and alcohols, e.g., ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agents, or emulsifying agents.
  • diluents such as water and alcohols, e.g., ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agents, or emulsifying agents.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricant, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscaramellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other preservatives, flavoring agents, and pharmaceutically acceptable disintegrating agents, moistening agents preservatives flavoring agents, and pharmacologically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a carrier, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base such as gelatin or glycerin, or sucrose and acacia.
  • Emulsions and the like can contain, in addition to the active ingredient, such carriers as are known in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can ' contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compounds can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2, 2-dimethyl-l, 3-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharmaceutically acceptable surfactant, such as soap or a detergent, suspending agent, such as carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
  • a pharmaceutical carrier such as a sterile
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Fatty acids can be used in parenteral formulations, including oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable salts for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, and alkyl pyridimium halides; anionic detergents such as dimethyl olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; polyoxyethylenepolypropylene copolymers; amphoteric detergents such as alkyl- ⁇ -aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and mixtures thereof.
  • suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, and alkyl pyridimium halides; anionic detergents such as dimethyl olefin sulfonates, alkyl, olefin, ether
  • Parenteral formulations typically contain from about 0.5 to 25% by weight of the conjugate in solution or suspension. Suitable preservatives and buffers can be used in these formulations. In order to minimize of eliminate irritation at the site of injection, these compositions may contain one or more nonipnic surfactants having a hydrophilic- lipophilic balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophilic- lipophilic balance
  • parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e.g., water, for injections immediately prior to use.
  • sterile liquid carrier e.g., water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • any number of assays well known in the art may be used to test whether a particular conjugate is sufficiently enzymatically active to convert alliin or a derivative thereof to allicin, and if this conjugate can successfully be directed to the desired site in vivo .
  • the dosage and frequency of administration is selected in relation to the pharmacological properties of the specific active ingredients. Normally, at least three dosage levels should be used. In toxicity studies in general, the highest dose should reach a toxic level but be sublethal for most animals in the group. If possible, the lowest dose should induce a biologically demonstrable effect. These studies should be performed in parallel for each compound selected.
  • the ID 50 level of the active ingredient in question can be one of the dosage levels selected, and the other two selected to reach a toxic level.
  • the lowest dose is that which does not exhibit a biologically demonstrable effect.
  • the toxicology tests should be repeated using appropriate new doses calculated on the basis of the results obtained. Young, healthy mice or rats belonging to a well-defined strain are the first choice of species, and the first studies generally use the preferred route of administration. Control groups given a placebo or which are untreated are included in the tests. Tests for general toxicity, as outlined above, should normally be repeated in another non-rodent species, e.g., a rabbit or dog. Studies may also be repeated using alternate routes of administration .
  • Single dose toxicity tests should be conducted in such a way that signs of acute toxicity are revealed and the mode of death determined.
  • the dosage to be administered is calculated on the basis of the results obtained in the above- mentioned toxicity tests. It may be desired not to continue studying all of the initially selected compounds.
  • Data on single dose toxicity e.g., ID 50 , the dosage at which half of the experimental animals die, is to be expressed in units of weight or volume per kg of body weight and should generally be furnished for at least two species with different modes of administration.
  • ID 50 the dosage at which half of the experimental animals die
  • compositions of the present invention are then ready for clinical trials to compare the efficacy of the conjugates to existing therapy.
  • a dose-response relationship to therapeutic effect and for side effects can be more finely established at this point.
  • the amount of conjugate of the present invention and of alliin to be administered to any given patient must be determined empirically, and will differ depending upon the condition of the patients. Relatively small amounts of the active ingredients can be administered at first, with steadily increasing dosages if no adverse effects are noted. Of course, the maximum safe toxicity dosage as determined should not be exceeded.
  • the present invention further provides a kit comprising in separate compartments a pharmaceutical composition containing the conjugate and an optional pharmaceutically acceptable carrier as described above, and a pharmaceutical composition containing alliin in an optional pharmaceutically acceptable carrier.
  • the kit also preferably contains instructions for administering the two compositions, including the dosage and the timing of administration of the two principles.
  • the alliin is preferably formulated for administration per os, intraparentally, or intravenously.
  • the present invention also provides a method for treating a disorder or disease which is treatable with allicin that comprises administering to an individual in need thereof a conjugate of the enzyme alliinase with a targeting carrier that targets the conjugate to a desired tissue or organ in the body, followed by administration alliin. In this way, allicin is generated at the desired tissue or organ, thus exerting its biological activity.
  • the targeting carrier is related to a cancer antigen
  • the method is suitable for treatment of cancer.
  • the targeting carrier is related to an antigen typical of an infectious disease
  • the method is suitable for treating the infectious disease.
  • the conjugate of the targeting carrier is administered first, followed by administration of alliin.
  • the alliin is administered from about 30 minutes to up about five hours after administration of the conjugate.
  • the administration of alliin may be repeated one or several times as necessary, in intervals to be decided according to the stage of the disease and condition of the patient.
  • Administration of alliin which is a non-toxic amino acid derivative, essentially potentiates the carrier-alliinase conjugate located on the target cell. The enzymatic activity of alliinase in the conjugate enables continuous generation of allicin at the target cell site. Generation of allicin depends on the availability of the substrate alliin.
  • Alliinase was purified from garlic cloves. 3- (2- Pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden) and dithiothreitol (DTT) from Sigma (St. Louis, MO, USA) .
  • SPDP Pyridyldithio propionic acid N-hydroxysuccinimide ester
  • DTT dithiothreitol
  • Activity assay of the alliinase-antibody conjugates was done by NTB methods either in cell free system (kinetics) or by ELISA, after adsorbing the conjugate to wells pre-coated with either anti-mouse antibodies or cells containing the antigen to the antibody. Binding of mAb-alliinase either to wells precoated with goat anti-mouse antibodies or to cell receptors (pre-fixed sub-confluent cells of N87 or CB-2 cells with 3% paraformaldehyde in PBS in 96-well plates) was done at room temperature for 1-2 hours. Unbound proteins were removed by washing (x3) with PBST (PBS containing 0.1% Tween 20).
  • Pegylation of alliinase was performed by adding various amounts of succini idyl carbonate polyethyleneglycol (SC-PEG) to the gel-filtered SPDP-alliinase in glycerol (10- 50%) in 50 mM phosphate buffer pH 6.5.
  • SC-PEG succini idyl carbonate polyethyleneglycol
  • the modification- mixtures were stored at 4°C overnight, then gel-filtered on SUPERDEX 200 (XK 16x70 cm) column, equilibrated with PBS, flow rate 1 ml/ in. Determination of polyethylene glycol attached to alliinase was done according to Nag et al (1996) .
  • Antibodies succini idyl carbonate polyethyleneglycol
  • mAb #N28.6 to ErbB-2 was partially purified from ascites fluid by using caprylic acid as described before (McKinney et al, 1987) or affinity chromatography on immobilized protein A or protein L.
  • N87 human gastric tumor cell line expressing the ErbB-2 receptors (described by Park et al, 1990) ; CB-2 cell line, generated by transfection of Chinese hamster ovary (CHO) cells with mammalian expression vectors that direct expression of ErbB-2 (Tzahar et al, 1996) ; and 32D cell line known to be devoid of the ErbB-2 receptors (described by Pinkas-Kramarski, 1996) .
  • CB-2 ErbB-2
  • DMEM/F12 (1:1) medium supplemented with antibiotics and 10% heat- inactivated BCS (bovine calf serum)
  • BCS bovine calf serum
  • FCS heat- inactivated FCS
  • the cell lines CB-2, N87 and 32D were kindly obtained from Prof. Y. Yarden and normal human foreskin fibroblasts were kindly obtained from Prof. B. Geiger, both from the Weizmann Institute. Cell Proliferation Assay
  • a stable conjugate was prepared by chemically coupling purified garlic alliinase with purified mAb to the
  • the SPDP- modified alliinase (1-3 residues SPDP/enzyme) was concentrated and stored in 25 mM phosphate buffer containing 50% glycerol and stored at -20 °C or coupled immediately to the reduced form of the SPDP-modified monoclonal antibody to ErbB-2 (mAb-SH) prepared as described below.
  • ErbB-2 mAb (1 mg/ml) was modified with SPDP (15 ⁇ l of 10 mM SPDP in DMSO/1.0 ml of mAb, 30 minutes, room temperature) , SEPHADEX G-50 gel-filtered as described above, concentrated and stored in PBS at 4°C (5-10 SPDP/mAb) .
  • the SPDP-mAb was reduced with DTT (5 mM) at room temperature for 10-20 minutes. Excess of DTT was removed by gel-filtration (PD-10) pre-equilibrated with 50 mM phosphate buffer pH 6.5 containing 10% glycerol. The protein containing fractions were collected and immediately combined with SPDP- alliinase, (10% excess of SPDP-alliinase over mAb-SH) . The conjugation mixture was stored 1 hour at 4°C in presence of 20% sucrose (added solid to the conjugation mixture) and concentrated by Centricon.
  • the conjugate alliinase-mAb was separated from free alliinase by SUPERDEX 200 gel-filtration, and then stored in 25 mM phosphate buffer pH 6.5 containing 50% glycerol and 2 mM pyridoxal 5-phosphate at -20°C.
  • Example 2 Effect of Allicin and Alliin on Tissue Cultured Cells
  • CB-2 cells were cultured in 96-well plate (2000 cells/well) for 6-16 h at
  • Example 3 mAb-Alliinase Conjugates Produce Allicin after Specifically Binding to Target Cells
  • CB-2 10,000 cells/well
  • N87 cells 20,000 cells/well
  • F (ab) -alliinase followed by the addition of alliin CB-2 cells (1,000 cells/well, 96-well plate) were grown for 6 hours at 37 °C in DMEM F12 medium with 10% iron-supplemented calf serum. Conjugate (F (ab) -alliinase) was added at various concentrations to the wells for 1 hour at 37 °C. The wells were washed x3 with the above medium and cultured for 16 hours with [ 3 H] -thymidine in the presence or absence of alliin (10 ⁇ g/well) . The plate was frozen at -20°C, trypsinized and the cells harvested. For each treatment triplicates were done.
  • Figure 5 shows that inhibition of cell proliferation, as determined by [ 3 H]- thymidine incorporation into living CB-2 cells upon treatment with conjugates consisting of F (ab) -alliinase followed by the addition of alliin, was dependent on the amount of conjugate bound to the cells .
  • Example 4 E fect of Bound mAb and mAb-Alliinase Conjugate on
  • the cancer cells used were N87 human gastric tumor cell line expressing the ErbB-2 receptors which have been described (Park et al, 1990) . These cells were cultured, harvested and used for the production of tumors in animals. [0144] The animals used were female CD1 (nude 5-7 weeks old) mice.
  • Radiolabeling of monoclonal antibodies was done with 125 I using the chloramine-T method (0.5 Ci Na 125 I/100 ⁇ g protein) according to (Hunter et al, 1962) .
  • the specific activity of free 125 I-mAb and 1 5 I-mAb-alliinase were 2.26 ⁇ Ci/ ⁇ g protein and 0.51 ⁇ Ci/ ⁇ g protein, respectively.
  • N-87 cultured cells (3-5 x 10 s ) were injected subcutaneously into the back of the mice. One to 5 days later, groups of 6 to 8 mice received either none or mAb-alliinase conjugate by intravenous injection (20 ⁇ g mAb/mouse) . Conjugates were injected with 3-4 days intervals.
  • mice were supplemented with Pyridoxine (vitamin B 6 ) in the drinking water (100 mg/L) . Every day mice were injected mtraperitoneally (IP) two times with alliin (0.2 ml of 15 or 30 mg/ml) with a 7 hours interval and once with Pyridoxal 5-phosphate (0.2 ml, 20mM in PBS/mouse) . Tumor size was measured every second day. Results
  • Example 5 Distribution and Clearance of Radiolabeled Anti- ErbB2 (Either as free mAb or inAb-Alliinase Conjugate) in Mice Containing N-87 Induced Tumors [0149] Tumors were generated in Female GDI nude mice by subcutaneous injection of human gastric tumor cells (10 x 10 s )
  • mice Two to three weeks later mice were injected intravenously with either 125 I-mAb anti-ErbB2 or 125 I-conjugated alliinase-mAb anti-ErbB2. At various time intervals mice were sacrificed and samples from various organs were analyzed for their 125 I radioactive content.
  • Example 6 Effect of Alliin Administration, in Mice Treated with mAb-Alliinase Conjugate, on Their Tumor Volume
  • Knipschid et all "Garlic, onions and cardiovascular risk factor: A review of the evidence from human experiments. Emphasis on commercially available preparations", Br J Clin Pharmacol 28:535-544 (1989)

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Abstract

On utilise des conjugués de l'enzyme alliinase avec un support protéique qui cible l'alliinase sur des cellules spécifiques en combinaison avec l'alliine pour produire l'allicine au niveau d'un site cible désiré. L'enzyme convertit l'alliine en allicine au niveau du site cible, tuant ainsi les cellules cancéreuses et les agents pathogènes.
PCT/US2001/049384 2000-12-26 2001-12-26 Generation in situ specifique d'un site de l'allicine au moyen d'un systeme de distribution cible d'alliinase pour le traitement des cancers, tumeurs, maladies infectieuses et autres maladies sensibles a l'allicine WO2002058624A2 (fr)

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AU2002246727A AU2002246727A1 (en) 2000-12-26 2001-12-26 In situ generation allicin for the treatment of cancer and infectious diseases
US10/451,849 US7445802B2 (en) 2000-12-26 2001-12-26 Site-specific in situ generation of allicin using a targeted alliinase delivery system for the treatment of cancers, tumors, infectious diseases and other allicin-sensitive diseases
IL15661701A IL156617A0 (en) 2000-12-26 2001-12-26 Conjugates of aliinase with a targeting carrier and pharmaceutical compositions comprising them
IL156617A IL156617A (en) 2000-12-26 2003-06-24 Conjugates of aliinase with an antibody and pharmaceutical compositions comprising them

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IL156617A (en) 2010-06-16
IL140555A0 (en) 2002-02-10

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