In Situ Allicin Generation Using Targeted Alliinase Delivery

Molecular Cancer Therapeutics 1295

Inhibition of tumor growth by a novel approach: In situ allicin generation using targeted alliinase delivery

Talia Miron, Marina Mironchik, David Mirelman, odorless precursor alliin (S-allyl-L-cysteine sulfoxide). Meir Wilchek, and Aharon Rabinkov Alliin is converted to allicin, pyruvate, and ammonia by the pyridoxal 5V-phosphate (PLP)-dependent enzyme allii- Department of Biological Chemistry, The Weizmann Institute of Science, nase (alliin lyase; EC 4.4.1.4) (3), which is enclosed in Rehovot, Israel compartments within the garlic clove cells (Scheme 1). Crushing the clove exposes alliin to the enzyme, to initiate the following reaction: Abstract Allicin (diallyl thiosulfinate), a highly active component in extracts of freshly crushed garlic, is the interaction product of non-protein amino acid alliin (S-allyl-L-cysteine sulfoxide) with the enzyme alliinase (alliin lyase; EC Allicin reacts rapidly with free thiol groups and 4.4.1.4). Allicin was shown to be toxic in various penetrates biological membranes with ease (4, 5). It mammalian cells in a dose-dependent manner in vitro. therefore disappears from the circulation within a few We made use of this cytotoxicity to develop a novel minutes after injection (2, 4–6). This explains why the approach to cancer treatment, based on site-directed versatile and valuable activities of allicin, including its generation of allicin. Alliinase from garlic was chemically potent antibiotic and cytotoxic effects, were demonstrated conjugated to a mAb directed against a specific tumor thus far only in vitro (2, 7–9). We present here a new marker, ErbB2. After the mAb-alliinase conjugate was approach to anticancer therapy based on a localized, site- bound to target tumor cells, the substrate, alliin, was directed production of allicin. This approach can also be added. In the presence of alliin, tumor-localized alliinase used to develop a wide range of antibiotic treatments. produced allicin, which effectively killed N87 and CB2, Alliinase is first targeted to the tumor. Conjugating the both ErbB2-expressing cells in vitro, whereas 32D cells (a enzyme to a carrier, in this case, a mAb specific to a tumor- murine hematopoietic progenitor cell line, devoid of the associated surface antigen, enables the targeting process. ErbB2 receptors) were not affected. Moreover, using N87, Alliin is then administrated into the circulation, which a human tumor cell line xenograft in athymic nude mice, results in the formation of allicin only at the site of alliinase we demonstrated for the first time, a high antitumor localization. The strategy for drug delivery based on activity of allicin that was produced in situ by the antibody-directed enzyme prodrug therapy (ADEPT) was conjugate, on alliin administration in vivo, while at the previously described (10–14). As in the ADEPT system, so same time other tissues were unharmed due to the inert alliinase-directed therapy uses mAbs specific to tumor cell nature of alliin and the high clearance rate of allicin. The antigens, to anchor the enzyme onto the cell surface. The effect of the treatment on tumor growth arrest became second step, however, consists of the administration of significant 2 weeks after its onset, and it continued to rise, alliin, a naturally occurring, inert non-protein amino acid reaching highly significant inhibition a week later. Ten that is converted to the cytotoxic allicin only on interaction days after the end of the treatment (day 18), tumor with the enzyme, anchored on the tumor cell surface. The growth inhibition was still the same. (Mol Cancer Ther. system presented here mimics the natural situation in 2003;2(12):1295–1301) crushed garlic cloves, where allicin formation occurs only after alliin becomes accessible to the enzyme. The advantages of this approach over the conventional ADEPT are the following: allicin and its precursor alliin Introduction which is nontoxic (unlike other drugs) are natural food Allicin (diallyl thiosulfinate) is the best known biologically constituents. Allicin acts swiftly as a powerful anticancer active component in the extract of freshly crushed garlic (1, agent and it has an extremely short lifetime. Addition- 2). Intact garlic cloves do not contain allicin, but only its ally, its secondary products such as allylmercaptoglutathione and allylmercaptocysteine were shown to bear beneficial antioxidant characteristics (4). Thus, a compre- Received 8/5/03; revised 9/18/03; accepted 10/1/03. hensive expression of the potent biological activity of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby allicin is achieved. marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods Grant support: Levine Center and Yeda Co. at the Weizmann Institute of Science. Materials Requests for Reprints: Aharon Rabinkov, Department of Biological 3-(2-Pyridyldithio) propionic acid N-hydroxysuccinimide Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-9344340; Fax: 972-8-9468256. ester (SPDP), DTT, and PLP were purchased from Sigma E-mail: [email protected] (St. Louis, MO). Alliin was synthesized according to

Ref. (15). Allicin was prepared by applying synthetic alliin (pH 6.5), containing 10% glycerol]. The protein was collected onto an immobilized alliinase column (16). Allicin concen- and concentrated by Centriprep-30 (Amicon, Beverly, MA) tration was determined as described in Ref. (17). 2-Nitro- at 4jC. The degree of SPDP-alliinase modification was 1–1.4 5-thiobenzoate (NTB) was synthesized according to Ref. (18). residues SPDP/E. The modified protein was stored in 50% [Methyl-3H]thymidine was purchased from Amersham glycerol at 20jC. SPDP modification of mAb: The only Pharmacia Biotech (Amersham, United Kingdom). difference from the procedure described above for alliinase Biochemical Analysis was a 20-M excess of SPDP over the mAb. The degree of Protein concentration was measured at 280 nm, using SPDP-mAb modification was 3–4 SPDP residues/mAb, and 1 1 0.1% the modified mAb was stored in PBS at 4jC. E280 = 77.000 M cm (E280 = 1.54) for alliinase and 1 1 0.1% 210.000 M cm for purified mAb (E280 = 1.4). The Preparation of the mAb-Alliinase Conjugate number of SPDP residues on the modified proteins was SPDP-mAb was reduced with DTT (5 mM, 10–20 min). determined according to Ref. (19). Purification of alliinase DTT was removed by gel filtration. Protein-containing was done according to Refs. (20, 21). Quantitative assess- fractions (mAb-SH) were collected and immediately com- ment of alliin and allicin was performed by high- bined with SPDP-alliinase, at a molar ratio of SPDP- performance liquid chromatography (17). Determination alliinase/mAb-SH (1.1/1). The conjugation mixture was of alliin content in the serum was done after i.p. injection of incubated 1 h at 24jC with 20% sucrose, adjusted to 50% alliin (0.2 ml of 30 mg/ml) to mice. Serum aliquots (25 Al) glycerol, and stored at 20jC. Before any further treatment, were treated with 90% methanol and proteins precipitated the conjugated alliinase was freed from sucrose and overnight at 20jC. After centrifugation, the supernatants glycerol by gel filtration. Further purification of conjugated were lyophilized. The dry material was dissolved in water proteins from non-conjugated was done by size-exclusion and was assayed for alliin according to Ref. (22). chromatography on Superdex G200 (1.6 60; Pharmacia) Enzymatic Activity with 50 mM phosphate buffer (pH 6.5), containing 10% Alliinase activity was determined by the NTB method in a glycerol and 2 mM PLP. cell free system according to Ref. (17). An ELISA system Radiolabeling of Proteins using 96-well plates was developed to assay the activity of Iodination of alliinase, free mAb, and conjugates was the enzyme conjugates. Wells were coated with either done with 125I, using the Chloramine-T method (0.5 mCi protein A (5 Ag/ml), or with paraformaldehyde-fixed N87 Na125I/100 Ag protein) according to Ref. (26). The specific or CB2 cells, at a sub-confluent culture density. Fixation was radioactivity of 125I-alliinase, 125I-mAb, and 125I-mAb- done with paraformaldehyde (3%) in PBS for 30 min, alliinase was 0.51, 2.26, and 0.85 ACi/Agprotein, followed by washing (3) with PBS. Binding of mAb- respectively. alliinase conjugates to the antigen was done at 24jC for Cell Lines and Tissue Culture 1–2 h in the presence of 2 mM PLP. After removal of N87, a human gastric adenocarcinoma cell line, express- unbound protein by washing (3) with PBS containing ing the ErbB2 receptors (27), HeLa HtTA-1 (28), BHK, baby 0.05% Tween 20, the activity of adsorbed conjugated hamster kidney cells, and NIH 3T3 cells were grown in 4 alliinase was determined by using NTB (3 10 M) and DMEM supplemented with antibiotics and 10% heat- 3 alliin (0.6 10 M)in50mM phosphate buffer (pH 7.2) inactivated FCS; CB2, a Chinese hamster ovary (CHO) cell containing 2 mM EDTA (0.1 ml/well). The decrease in line transfected with mammalian expression vector ErbB2 A412 nm was recorded after 30 min. Wells, to which no (29), was grown in DMEM/F12 (1:1) medium supple- enzyme was added, served as controls (17). One unit of mented with antibiotics and 10% heat-inactivated bovine activity was defined as the amount of enzyme required to calf serum; 32D, a murine hematopoietic progenitor cell produce 1 Amol of pyruvate per minute. line (30), devoid of the ErbB2 receptors, was grown in Antibodies suspension in RMPI 1640 supplemented with glutamine, N28 (IgG1) mAb, anti-ErbB2 (23, 24), was purified from antibiotics, IL-3, and 10% FCS (31). ascites fluid by using caprylic acid (25) or by affinity Cell Proliferation Assay 3 chromatography on immobilized protein A. F(ab)2 was [Methyl- H]thymidine (Amersham) incorporation into prepared from N28, by pepsin digestion (1 mg pepsin/15 DNA was measured in 96-well plates. All the experiments mg antibodies) in 0.1 M citrate-phosphate buffer (pH 3.7), performed with cells were carried out in triplicates. CB2 or for 4 h at 37jC, followed by Superdex-200 (XK16x70) size N87 adhesive cells (1,000–20,000 cells/0.1 ml/well) were xtl exclusion chromatography in PBS. HCV-AB 68, a purified grown at 37jC for 6 h after seeding. Growth medium was human mAb against the E2 protein of the hepatitis C virus, replaced by fresh medium containing 2 mM PLP. Free mAb was used to prepare a non-relevant control conjugated with or mAb-conjugate was added to the cells for 1–2 h. Cells alliinase. were then washed (3) with medium containing 0.4 mM PLP Protein Modifications and resupplied with growth medium containing 0.02 mM SPDP modification of alliinase: SPDP (50 mM in ethanol) PLP. 32D cells were grown in suspension in 12-well plates was added at a 5-M excess to alliinase in 25 mM Na phosphate (200,000 cells/ml). Treatment with mAb or conjugates (pH 6.5), containing 50% glycerol for 1 h at room proceeded as described above. Unbound proteins were temperature. Excess SPDP was removed by gel filtration washed off with medium containing PLP by centrifugation [Sephadex G-25 equilibrated with 50 mM phosphate buffer (1200 rpm, 5 min). Treated cells were resuspended with fresh

medium and reseeded in 96-well plates (20,000 cells/well). different concentrations. The extent of CB2 cell proliferation From this stage on, both adhesive and non-adhesive cells in the presence of allicin was evaluated by following were supplemented with [methyl-3H]thymidine (0.6– [3H]thymidine incorporation after incubating both for 16 h 0.8 mCi/well) and incubated in the presence or absence of at 37jC. Cells were harvested and the amount of [3H]thy- 3 alliin (0.6–1.2 10 M). After 16 h incubation at 37jC, plates midine incorporation was determined (Fig. 1). The results were frozen (20jC, 1 h), and harvested following trypsin demonstrate that pure allicin inhibits DNA synthesis in a treatment. Cell viability was determined with trypan blue dose-response manner; 50% inhibition is obtained at about (0.025%) for 10 min in 24-well plates. 13 AM and 90% inhibition at about 25 AM. A dye exclusion Animal Experiments test corroborated the above results. CB2 and N87 cells were All animal studies and protocols were approved by the incubated for 16 h in the presence of alliin or allicin, and Weizmann Institutional Animal Care and Use Committee viewed under a light microscope after staining. Lethality (WIACUC). was observed at allicin concentrations of about 20–30 AM, A Xenograft Mouse Model whereas alliin was nontoxic at concentrations up to 200 M. Preparation of the Conjugate mAb ErbB2-Alliinase Tumors were generated in Female CD-1 nude mice (6 weeks old) by s.c. injection of N87 (3–5 106 cells/ Covalent conjugates of anti-ErbB2-alliinase were pre- mouse) into the back of the animals. Three to 5 days after pared by binding the enzyme to the anti-ErbB2 or to its tumor cell implantation (early tumors), mice were grouped F(ab)2 derivative, yielding 1–2 alliinase/mAb (mole/mole). (six to eight/group) according to mode of treatment and The conjugates of anti-ErbB2-alliinase maintained their subsequent alliin administration (+/): (1) PBS/; (2) target recognition of live and fixed tumor cells as well as PBS/+; (3) anti-ErbB2 (N28)/+; (4) anti-ErbB2-alliinase their alliinase activity, as determined by allicin formation conjugate/; (5) anti-ErbB2-alliinase conjugate/+; (6) allii- (Fig. 2A). The activity of the conjugates bound to fixed cells nase/+; (7) HCV-ABxtl68-conjugate/+. All the mice were was concentration dependent (Fig. 2B). constantly supplemented with pyridoxine (vitamin B6) in Effect of Alliinase Conjugates on Cells in Vitro their drinking water (100 mg/l). PLP was injected i.p. We used anti-ErbB2 to establish a general approach of (0.2 ml, 20 mM in PBS/mouse) 30 min before each i.v. targeted cell killing, employing N87 (a human gastric tumor injection of the alliinase conjugate or its controls. Anti- cell line) and CB2 (a Chinese hamster ovary cell line) ErbB2, anti-ErbB2-alliinase conjugate, and HCV-ABxtl68- expressing ErbB2 receptors, and exploiting the fact that this alliinase were used at 20 Ag mAb/mouse, and alliinase monoclonal anti-ErbB2 is not internalized after binding to control at 20 Ag/mouse. Animals receiving all modes of its surface receptor (24). However, on binding to the cell, treatment and controls were injected 5 at 3- to 4-day this antibody enhances cell proliferation at a rate of about intervals. Alliin (0.2 ml of 30 mg/ml) was injected i.p. into 10–20% (24). Therefore, the suppression obtained by groups 2, 3, and 5–7 at a 7-h interval, twice a day, during 2–4 weeks. Tumor volume was measured every second day, by using calipers. Mean tumor volume of the different groups was compared by using variance analysis. Due to NIH guidelines and the Animal Welfare Act, mice were sacrificed as soon as the tumor reached 1.5 cm in volume. Organ Distribution of 12 5I-mAb-Alliinase Conjugate This study was carried out with N87-treated nude mice 3 weeks after tumor cell implantation. Mice were injected i.v. with either 125I-alliinase, 125I-anti-ErbB2, or 125I-anti- ErbB2-alliinase conjugate (1 106 cpm/mouse). At various time intervals, two mice were sacrificed; organs were excised, washed with PBS, weighed, and their 125I content was determined. Statistical Analysis Results are shown as the mean values F SD. The in vitro results are average data of triplicate experiments. The in vivo experiments with anti-ErbB2 and anti-ErbB2- alliinase and PBS have been performed at least twice with similar results. ANOVA and Tukey test were used to ana- lyze statistically significant differences between the groups. Figure 1. Inhibition of CB2 cell proliferation by allicin. After seeding, CB2 cells were grown in culture in 96-well plates (2000 cells/well) at 37jC for 4 – 6 h. Pure allicin was added to final concentrations ranging between Results 3 0 and 54 mM, followed by [ H]thymidine (0.8 mCi/well). Cells were Effect of Allicin and Alliin on Cells inTissue Culture harvested after an incubation period of 16 h and the level of incorporated [3H]thymidine was determined. The 100% incorporation value was The effects of the inert substrate, alliin, and the active obtained from non-treated control cells. The mean of the [3H] incorporation product, allicin, on CB2 cells in culture were determined at (%) F SD is presented.

CB2, we found that the former exhibited a certain degree of conjugate recognition (5–15% of the latter). Therefore, we had to resort to a non-adherent cell line, 32D, devoid of ErbB2 receptors (31). Thymidine incorporation resulting from the specific binding of the conjugate to N87 and to 32D cells was examined. Cells were treated with either anti-ErbB2 or with alliinase-anti-ErbB2 conjugate, in the

Figure 2. Determination of enzymatic activity of mAb-alliinase conjugates after binding to cancer cells expressing ErbB2 receptors. A, sub- confluent CB2 and N87 cell cultures in 96-well plates were fixed with paraformaldehyde, and treated with either mAb anti-ErbB2, or with each of the conjugates mAb anti-ErbB2-alliinase and F(ab)2 anti-ErbB2-alliinase (antibody concentration, 2 Ag/well). Enzymatic activity was determined by allicin generation as described in the experimental protocols after careful washing of wells from unbound material. The mean decrease in A 412 nm F SD is given. DA 412 nm =[A 412 nm non-treated] [A 412 nm treated with alliin]. CB2 (n), N87 ( ). B, alliinase activity as a function of conjugate bound to cells. A sub-confluent CB2 culture was treated as above. Growing amounts of mAb or mAb-alliinase were added to the cells. Results are expressed as DA 412 nm. mAb anti-ErbB2 (n), mAb anti-ErbB2-alliinase ( ).

Figure 3. Inhibition of [3H]thymidine incorporation. A, inhibition of targeted alliinase-anti-ErbB2 in the presence of alliin must [3H]thymidine incorporation into CB2 cells on treatment with the be compared to that without alliin, rather than to a PBS conjugate F(ab)2-alliinase, followed by the addition of alliin. After seeding, control. Binding of the anti-ErbB2-alliinase conjugate to CB2 cells (10,000 cells/well, 96-well plates) were grown at 37jC for 6 h. ErbB2-expressing cell monolayers (CB2) did not impair The conjugate F(ab)2 anti-ErbB2-alliinase was added to the cells at various concentrations in the presence of 2 mM PLP, for 1 h. Cells were then the accessibility of alliin to alliinase, and cell proliferation washed (3) with medium containing 0.4 mM PLP, resupplied with fresh was inhibited on addition of the substrate. The inhibition growth medium, and incubated for 16 h with [3H]thymidine in the of [3H]thymidine incorporation in CB2 was dose depen- presence of alliin (10 Ag/well; ) or in its absence (5). B, inhibition of [3H]thymidine incorporation in 32D and N87 cells on the following dent (Fig. 3A). In the search for a cell line that does not treatments: none, mAb anti-ErbB2, and the conjugate mAb anti-ErbB2- respond to anti-ErbB2 conjugate, we examined several alliinase in the presence of added alliin or in its absence. N87 and 32D cell adherent cells such as HeLa (human), BHK (hamster), and cultures were treated with mAb anti-ErbB2 or with mAb-anti-ErbB2- alliinase conjugate (antibody concentration, 1 Ag/10,000 cells; alliinase NIH-3T3 (murine). We have screened these cells for the activity of the conjugate, 0.15 unit/10,000 cells) for 1 h at 37jC. After presence of ErbB2 receptors. After cell treatment with the unbound protein was washed away, [3H]thymidine (0.6 ACi/well) was conjugate, two techniques were used to assess its effects: added to the cells, followed immediately by the addition of alliin (10 Ag/ 3 determination of alliinase activity on paraformaldehyde- well). [ H]thymidine incorporation into the DNA was determined. 32D ( ) and N87 (n) in the presence of alliin; 32D (5) and N87( ) in the absence fixed cells, as well as of thymidine incorporation in live of alliin. [3H]thymidine incorporated without any treatment served as the cells. Comparing the above three cell lines to N87 and 100% value. The mean percent [3H] incorporation F SD is presented.

presence and absence of alliin. Inhibition of [3H]thymidine incorporation in N87 treated with the conjugate in the presence of alliin was higher than 70%, while no inhibition was observed in 32D cells under the same conditions (Fig. 3B). N87 and CB2 cells, treated with mAbN28-alliinase and alliin for 16 h, lost viability as observed from a dye- exclusion test (data not shown). It is worth noting that cells treated with anti-ErbB2 (mAbN28) or anti-ErbB2 alliinase conjugates in the absence of alliin, show accelerated tumor cell growth as previously described (24). Distribution of Anti-ErbB2-Alliinase Conjugate in a Xenograft Mouse Model The ability of the anti-ErbB2-alliinase conjugate to bind selectively to tumor cells in vivo was evaluated by using 125I-labelled anti-ErbB2-alliinase in mice injected s.c. with human N87 tumor cells. Due to the virulent nature of N87 cells, and their quick proliferation, the time schedule of inhibition experiments and distribution experiments was different. Whereas a large enough time interval was needed to monitor tumor growth and the model studied was an early tumor, the distribution could be performed on mature tumor. Two to 3 weeks after tumor implantation, mice were injected i.v. with the conjugate 125I-anti-ErbB2-alliinase, or with its separate two components, 125I-anti-ErbB2 and 125I-alliinase. The bio-distribution of 125I in various organs of the treated mice after 24 h is shown in Fig. 4. After 125I-anti-ErbB2-alliinase injection, there is a specific accumulation of 125I-labeled protein in the tumor, as compared to other organs. Maximal accumulation of the conjugate occurred after 24 h, and its half-life in the tumor was about 72 h. However, the half-life of the conjugate in the blood was less than 8 h. A similar bio-distribution pattern was Figure 4. Bio-distribution of 125I-anti-ErbB2-alliinase, 125I-anti-ErbB2, observed for the 125I-anti-ErbB2-treated mice, but the half- and 125I-alliinase in organs of nude mice with tumors originating from life in the tumor was around 5 days (half-life in the blood N87 cells. A, bio-distribution of radiolabeled conjugate or its control components. Mice (two/group) were injected i.v. with the radiolabeled was less than 8 h, as in the case of the conjugate). conjugate or its control components (106 cpm/mouse) and sacrificed 24 h 125 I-labelled alliinase did not accumulate in the tumor later to determine tissue uptake. Organs were excised, weighed, and (Fig. 4). The half-life of alliin in the serum was about 4–5 h; counted. mAb anti-ErbB2, mAb anti-ErbB2-alliinase n, alliinase . Tissue uptake was calculated as the mean percentage of the injected dose per therefore, it was administered twice a day. gram of tissue. B, time dependence of 125I-anti-ErbB2-alliinase bio- Tumor Treatment with mAb-Alliinase Conjugate, Fol- distribution in organs of nude mice bearing tumors originating from N87 lowed by Successive Alliin Administrations cells. Mice (two/group) were injected i.v. with the radiolabeled conjugate (106 cpm/mouse) and sacrificed after 8 ,24 , and 72 h. Organ The effect of the mAb-enzyme conjugate, with and samples were weighed and counted. without subsequent alliin administration, on tumor growth in vivo was studied using a human xenograft in mice. An early model was used, in which treatment with conjugates controls supplemented with alliin were tested, and none started 3–5 days after tumor cells were injected. The F suppressed tumor growth: alliinase alone and alliinase control group treated with PBS ( alliin) represents the non- conjugated to a nonrelevant mAb (HCV-ABxtl68) (groups 6 treated baseline. The rate of tumor growth, in mice treated and 7, respectively). Alliin itself had no effect on tumor size with the anti-ErbB2-alliinase conjugate, followed by suc- as seen from control mice (PBS, groups 1 and 2). cessive administrations of alliin (group 5), slowed down significantly (P < 0.001) as compared to alliin-supplemented control mice that were not treated with conjugate Discussion (PBS). Other controls that did not suppress the kinetics of Since its discovery in 1944, allicin has been considered as a tumor growth were anti-ErbB2 (N28) alone and the anti- potent antimicrobial agent (1). A decade after its discovery, ErbB2-alliinase conjugate, without subsequent alliin ad- its antiproliferative and cytotoxic activities started to be the ministration (groups 3 and 4). These controls caused an object of keen interest (32). However, allicin was shown to even somewhat enhanced tumor growth, which actually be unstable and short-lived. Furthermore, after injection, its magnifies the effect of the treatment (Fig. 5). To test the fast disappearance from the circulation thwarted scientists high specificity of targeted allicin production, two more from attempting to use it as a chemotherapeutic agent.

with its short life duration in the body; and (d)the conversion of excess allicin into beneficial derivatives such as allylmercaptocysteine and allylmercaptoglutathione that possess antioxidant and SH-modifying activities (4). Because the animal model imposes some ethical restric- tions, our results endorse, at least for the moment, allicin treatment as a supplementary therapy to surgery and as a preventive of metastases. This pioneering attempt to use targeted-allicin production in cancer therapy will be followed by further experiments that should investigate the potential of this approach. Various tumors and their related surface recognition molecules will be examined. In addition to growth inhibition of any type of cancer cell, the targeted-alliinase principle can be used to eliminate pathogens. The only requirement is the availability of highly specific mAbs, or other kinds of target-specific carriers. Ligation of such carriers to alliinase can be done by using chemical, or recombinant fusion methodology. Figure 5. The effect of targeted-alliinase/alliin treatment on tumor volume. Tumors were induced in nude mice with N87 cells. Mice (six to eight/group) were treated with the conjugate mAb anti-ErbB2-alliinase, Acknowledgments with (.) or without (o) subsequent alliin administration (+/). Control The mAb N28 to ErbB2 receptor was kindly provided by Dr. E. Hurwitz; experiments with either PBS (E), anti-ErbB2 (n), alliinase (4), or HCV- CB2, N87, and 32D cell lines were kindly obtained from Prof. Y. Yarden of ABxtl68-alliinase-nonrelevant conjugate ( ); were followed by alliin the Weizmann Institute Rehovot, Israel. The mAb HCV-ABxtl 68, a purified administration. Either conjugate (relevant and nonrelevant) as well as free human mAb against the E2 protein of the hepatitis C virus, was kindly enzyme, free antibodies, and PBS were given twice a week, i.v. with 3 – 4 provided by XTL Biopharmaceuticals Ltd., Nes Ziona, Israel. days interval, five treatments for each group. The measurements were continued for two more weeks. Alliin (240 mg/kg) was administered twice a day (i.p. injections). Tumor volume was monitored. Comparison of the References average tumor volume of mice treated with anti-ErbB2 alliinase/+ to all the 1. Cavallito, C. J. and Bailey, J. H. Allicin, the antibacterial principle of other treatments (after 2 weeks of treatment) was significant (P < 0.001) Allium sativum. I. Isolation, physical properties and antibacterial action. by ANOVA. J. Am. Chem. Soc., 66: 1950 – 1951, 1944. 2. Koch, H. P. and Lawson, L. D. Garlic: The science and therapeutic application of Allium sativum l. and related species, second edition, p. 329. 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Talia Miron, Marina Mironchik, David Mirelman, et al.

Mol Cancer Ther 2003;2:1295-1301.

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