CA2424255A1 - Immunotoxins - Google Patents
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Abstract
Ep-CAM is a tumor-associated antigen overexpressed in many solid tumors but shows limited expression in normal epithelial tissues. To exploit this favorable expression pattern for targeted cancer therapy an Ep-CAM-specific recombinant immunotoxin was developed and its antitumor activity investigated. The immunotoxin 4D5MOCB-ETA was developed by genetically fusing a truncated form of ETA (ETA252-608KDEL) to the highly stable humanized scFv 4D5MOCB.
Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts. Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen binding affinity. In vitro, 4D5MOCB-ETA
potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC50 values ranging from 0.005 to 0.2 pM. Upon systemic administration in mice,4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumorblood ratio of 5.4. The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon or squamous cell carcinomas
Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts. Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen binding affinity. In vitro, 4D5MOCB-ETA
potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC50 values ranging from 0.005 to 0.2 pM. Upon systemic administration in mice,4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumorblood ratio of 5.4. The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon or squamous cell carcinomas
Description
Immunotoxin FIELD OF THE INVENTION
The present invention relates to the field of cancer therapy and particularly to targeted therapy using immunotoxms.
BACKGROUND OF THE INVENTION
The lack of significant advances in the treatment of metastatic or refractory cancers has stimulated the design of novel approaches to targeted cancer therapy such as the use of antibody~~
based cancer therapeutics Despite favorable initial responses, most advanced solid tumors develop resistance to standard treatments and relapse as incurable metastatic diseases ( 1 ). Since increasing the dose of conventional anticancer agents results in unacceptable side effects, the design of novel therapies based on the use of tumor selective targeting ligands and effector domains employing different mechanisms of action is of great rmportance. Antibodres targeting tumor-associated antigens and equipped with intrinsic cytotoxic or rmmunostimulatory effector functions have shown promising antitumor activity in preclinical and clinical studies (2-4).
Because of their different mechanism of action and especially because of their built-in targeting function that conventional anti-cancer agents do not have, immunotoxins may add new options for the treatment of malignancies resistant to conventional treatments (26,28). ETA and its homologues irreversibly block protein synthesis in cells by ADP-ribosylating a posttranslationally modified histidine residue of elongation factor 2, called diphthamide, which ultimately triggers apoptosis (48). Although resistance of°cells to ET'A was described as a consequence of the mutation of the crucial histidine residue or loss of enzyme activity required for diphthamide synthesis (49,5(1), this is a rather uncommon event and has not been confirmed in other cell systems. Nevertheless, the loss of the tumor antigen used for uptake of the antibody-toxin tizsion protein by the tumor cell is another conceivable mechanism of resistance.
An immunotoxin is a chimeric protein in which a toxin moiety is chemically or genetically linked to a monoclonal or recombinant antibody Antibodies with speciticities for various tumor-associated antigens have been investigated as carriers of"toxins, and the majority of those that target solid tumors have employed the effector function ot'truncated ET'A
(.ETA) which lacks the cell binding domain I (aa 1-252) (20), F.T A irreversibly inhibits protein synthesis by ADP-ribosylation caf elongation factor 2 and therefore has to gain access to its intracellular target in tire Immunotoxin cytoplasm (20,21 ). Thus, the most promising antigens for immunotoxin therapy are those that are efficiently internalized into tumor cells upon antibody binding by receptor-mediated endocytosis (22-25 ).
A number of chemical and recombinant immunotoxins that employ either plant or bacterial toxins as eff'eetor domains and that target distinct cell surface antigens associated with tumor cells have been shown to be potent and selective anti-cancer agents in preclinical studies (28). However, only few of them proved to be promising candidates for clinical use.
Several immunotoxins, either as chemically~linked first generation or recombinant second generation formats, have been tested in animal models and in patients with advanced solid tumors (20,26). Although early clinical data using imrnunotoxrn therapy for refractory tumors remains promising, the induction of neutralizing antibodies and dose-limiting side effects associated with VL,S or liver toxicity still remain obstacles to effective therapy (27-29). To overcome these limitations, more promising tumor-associated antigens have to be validated as targets for nc:w irnmunotoxin generations which are equipped with rationally engineered effector functions (2,30,31 ).
Mayor responses have been reported so far only for leukemias. In phase I
studies using LMI3-2, an FTA based scFv immunotoxin which targets lL-2 receptor p-chain, responses have been observed treating hematologic malignancies (51,52). Moreover, two thirds of the patients with refractory hairy-cell leukemia involved in a phase I study showed complete remission after treatment with 81.22, a recombinant F.'fA-based dsFv immunotoxin specific for the CD22 surface antigen (53)0 Both immunotoxins showed minor or reversible toxic side effects and thus merited to be involved in phase I studies for which currently a 'larger number of patients are being recruited.
T wo recombinant F;TA-based dsfv immunotoxins are also currently being evaluated for the treatment of advanced solid tumors. The immunotoxin SSI(dsFv)-PE3R is directed against cells expressing mesothelin, a protein normally produced by mesothelial cells and expressed also on malignant mesotheliomas and ovarian carcinomas (54). The immunotoxin LMB-9 has been derived from the monoclonal antibody B3 that targets the LewisY antigen (55), which is also widely expressed in epithelial tissues, a fact that contributes to safety concerns. LMB-9 and SS I (dsfv)-PE3R are currently being tested in phase 1 clinical trials in patients with advanced solid tumors and first results are eagerly awaited. In a previous study with 3H patients suffering from advanced carcinomas the chemical conjugate of~monoclonal antibody B3 and ETA induced one complete and one partial response (S6). In these studies vascular leakage due to capillary lmmunotoxin 4 damage was found to be dose-limiting, and subserauent preclinical investigations revealed significant binding of the B3 antibody to LewisY expressed on endothelial cells (57).
In addition to HER-2/neu (25), Ep-CAM represents another more promising target for antibody-based therapy of solid tumors due to its abundant expression in many carcinomas and its limited distribution in normal epithelial tissues (5). Although, Ep-C.AM expression is not exclusively restricted to tumor cells, Riethmuller and co-workers found that application of the anti-Ep-CAM
monoclonal antibody 17-1 A in patients with resected colorectal carcinoma or minimal residual disease reduced the overall mortality by 32%, decreased the recurrence rate by 23% (1), and reduced the number of distant metastases (8).
Ep-CAM t is a 40 kDa transmembrane protein overexpressed in many solid tumors including carcinomas of the lung, breast, ovary, colorectum and squamous cell carcinoma of the head and neck (5). The limited expression of Ep-c"AM in normal epithelial tissues (5,6) makes this antigen an attractive target for cellular and antibody-based immunotherapy (7-9).
Recently, a transgenic mouse model mimicking the Ep-(:SAM expression pattern in humans further validated the suitability of this target for imrnunotherapy by showing no localization of the monoclonal antibody MfW'31 in Ep-CAM-positive ruormal tissues ( 10).
The role of Ep-('AM in carcinogenesis and malignant progression is still unclear, but there is increasing evidence that it modulates cell-cell interactions ( 1 1 ) and that its expression correlates with the rate of cell proliferation ( 12) In addition, a promoting role of Ep-CAM in tissue invasion and metastasis has been suggested ( 13), and a strong correlation between Ep-CAM expression and tumor progression has been found in patients with squamous cell carcinoma of the head and neck'. Ep-C.'AM-specific antibodies have been used in imaging studies to detect primary tumors and localize distant. metastases in patients mth SCL,C (14) and NS("LC (15), to trigger antitumor immune responses ( 16), and to deliver cytotoxic effector molecules to tumors in preclinical models ( 17,18) and in patients ( 19).
~ The abbreviations used are: Ep-C'AM, epithelial cell adhesion molecule;
S('LC, ,mall cell lung cancer; NSC'L.C, non small cell lung cancer; ETA, P.ceudvmona.v aemn,s;lnacu exotoxin A; VL..S, vascular lean syndrome; seFv, single chain antibody fragment: dsFv, disulfide-stabilized ,ingle-chain antibody fragment;
FBS, fetal bovine serum; IMA(', innnobilized ion-metal affinity chromatography.; PAGE, polyacryiamide gel electrophoresis; PBS, phosphate buffered saline; MT'f, i-14,5-dimethylthiazol-?-yl]-?,5-etiphenyltetrazolium bromide;
F1TC' fluorescein isothiocyanate, MFI, mean fluorescence intensity; E(.iF, epithelial growth factor AI.T, alanme aminotransferase; AST, aspartate aminotransferase; TNF, tumor necrosis factor D. Tschudi, unpubLshed observation.
lmmunotoxin Although in a previous study we reported the ability of the chemically conjugated immunotoxin lYIO('31-ET'A to eradicate small tumor xenografts in mice as well as its failure to delay the growth of larger tumors ( 18), we concluded that, due to its relatively large size of 200 kDa, the immunotoxin was unable to homogeneously distribute wrthin the tumor mass and thus could only affect an insufficiently small proportion of clonogenic tumor cells. Support for this hypothesis is promded by others who reported an inverse correlation between immunotoxin size and efficacy (32j. The tumor targeting and tissue distribution properties of ammunotoxins can be substantially improved by using small scFv as targeting ligands (22,2ni,25,3:3-37). We have recently described the enhanced tumor localization of scFv 4DSM0('.B, which was derived by grafting the hypervariable loops of monoclonal antibody MO('3 l onto the humanized framework of'the anti-HF:R-2ineu scl~'v 4DS and by additionally changing eight critical care residues to obtain a high molecule stability (3H). We have now teound that the development ofa fully recombinant Ep-t."AM-specific single-chain immunotoxin based on 4DSMOCB, can be employed to achieve favorable tumor localization and potent antitumor activity against carcinomas of diverse histologrcal ongins in vivo.
SUMMARY OF THE INVENTION
The present invention is predicated on the finding that 4DSMO CB-ETA exerts significant growth inhibition upon systemic administration to mice bearing large established tumor xenografts ( 160 mm;' from colorectal, small cell lung or squamous cell carcinoma of the head and neck. The present invention reports on two dose schedules that were well-tolerated and proved to be very effective in inhibiting tumor growth. The three-week treatments with a total dose of 45 trg eradicated a significant fraction of°the tumors, and some mrce remained tumor-free during the whole study. However, in contrast, after completion of the shorter one week treatment with a total dose of 30 lrg 4DSMOCB-ETA, HT29 tr.unors rapidly resumed their growth.
BRIEF DESCRIPTION OF THE DRAWINGS
Thz present invention is illustrated but not limited by the attached drawings of which:
Frgure l A is a schematic representation of the scFv-toxin fusion protein precursor, which includes the ompA signal sequence for periplasmic expression. 'The scFv antibody fragment ~4D~M()(~B is fused to the Pseudomonas exotoxin A (ET'A;52_~os) by the linker shown. The protern is tlanked by two hexahiscidine tags, the C.'-terminal of' which precedes the ER retention signal 1CDEL.
lmmunotoxin 6 Figure ( B is a three-dimensional model of the mature 4DSMOCB-ETA., The structure of the scFv (VL in red, VH in orange), of ET'A;52..~r,x (domain II in light-blue, domain Ib in green and domain III in violet) and of the linking peptide (green) are shown. Both hexahistidine tags are indicated in yellow.
Figure 1 (.' is a copy of a chromatogram. 'Total extract of SB536 bacterial culture samples before (-) and after (+) IPTG induction and 10 p,g of 4DSMOCB-ETA immunotoxin purified by Ni2+-IDA and anion exchange affinity chromatography columns coupled in series, were analyzed on 10% SDS-PAGE under reducing conditions Figure I D shows the immunotoxm proteins present in the same samples as visualized on a Western blot using a HRP-conjugated anti-tetrahistidine antibody. Markers are shown in lane M:
myosin ( M~ 20H,000), w-galactosidase (Mr 1 19,000), bovine serum albumin (M,.
94,000), ovalbumm (Mr 51,100), carbonic anhydrase (Mr 35,400) and soyabean trypsin inhibitor (M~
2H,so0) Figure 2 shows the chromatograms before (0 h) and after 2 h, 4 h, H h, 10 h and 20 h incubation were recc'~rded at 2H0 nm. The monomers eluted at approximately l.4 ml as verified by calibration with the molecular weight standards alcohol dehydrogenase (A, M~ I 50,000), bovine serum albumin (B, M, H6,000) and carbonic anhydrase (C', M,29,000). which eluted at 1.31 ml, 1.38 ml and 1.54 ml, respectively (retentiun volumes shown by arrows). The stability of 4D5MOCB-ETA was assessed by size exclusion chromatography. 'The immunotoxin was incubated at a concentration of 200 ugiml at 37"C: in PBS and samples were analysed by gel filtration at different time points for comparison.
Figure 3 is a graphic depiction of cell growth measured in MTT assays as described in the "Material anc~ Methods" section. Data represent mean values of at least six independent determinations each carried out in quadruplicates (overall SD<5'%). Four Ep-(:AM-positive tumor cell lines were incubated for 72 h with 4DSMO('B-ETA at concentrations ranging from 0.0001 to 100 pM.
Figure 4 is a graphic representation of impairment of liver function upon treatment with 4DSMOC'B-ETA. C'S7BL/6 mice were treated every other day with escalating doses of 4D5MOC'B-ETA Two groups received 5 pg (?50 pg.kg-l ) or 10 pg (500 pg.kg-1 ) doses for three cycles, while another group was treated twice with 20 yg ( 1000 ltg.kg-1 ).
Twenty-four hours after Immunotoxin the last challenge the activities of plasma transaminases were determined and compared to mice treated with PBS (0 pg.kg-1 ). The transaminase activities of mice treated with a single lethal dose of wild-type E'TA- (85 pg.kg-1), as described by Schumann et al. (47)., are also shown. Data are expressed as the mean t SD (n = 3).
Figure 5 is a graphic representation of tumor growth illustrating the antitumor effect of ~DSMOCB-ETA in mice. Athymic mice bearing large tumor xenograf'ts ( 160 mm; in average) derived from the Ep-CAM-positive cell lines HT29, SW2 and (.'AC.27 remained untreated ( ? -) or were treated by i.v. injections every second day with either nine doses of 5 pg 4DSMOCB-ETA each for three weeks (-; -), or with three doses of 10 tig each (-'? -).
In a control experiment, mice bearing Ep-CAM-negative (.'OLO320 xenografts were also treated with 4DSMOCB-E.TA
according to the dose schedules mentioned above. 'The tumor size is given relative to the initial median tumor size of 160 mm3 at the start of treatment and data represent the mean values pSD
of'the various groups (n - 7).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The immunotoxin 4DSMOC:B-ETA was developed by fusing the highly stable humanized scFv 4DSMO('B (38) to a truncated form of hTA comprising amino acids 2.52-608 and the C-terminal eukaryotic ER retention sequence KDEI.. The 4DSMOCB-ETA is extremely potent in the femtomolar range and potently inhibits the growth of°carcinoma cells of diverse histological origins in a highly antigen-specific manner as demonstrated by an increase in the cytotoxic potency by more than four orders of magnitude, compared to antigen-negative cells. Moreover, the antigen-specific action of 4DSMOCE~-ETA was further corroborated in competition assays using an excess of scFv 4D5M0('B. Similar to the findings reported from immunotoxins targeting other tumor-associated antigens including HER-2/neu and E(iF
receptor (25,59), the cytotoxic activity of 4D5M0(:B-f=TA did not correlate with the amount of target antigen expressed on the tumor cell surface. Thus, it is likely that other cell type-specific parameters, such as rate of internalization, intracellular trafficking and fate of the enzyme domain are further determinants of immunotoxin efficacy. In terms of its in vitro cytotoxicity 4DSMOCB-ETA is the most potent Ep-C'AM specific iinmunotoxin that has been reported in the literature and was 1000-fold more potent than the chemical immunotoxin conjugate MOC'31-E:TA ( 18).
~, prerequisite for the optimal binding of antibody-based therapeutics to target antigens expressed an the surface of tumor cells and for efficient tumor localization is protein stability under physiological conditions. The extremely potent cytotoxicity shown by 4D5MOCB-ETA may at lmmunotoxin least partly be due to the stability of the targeting scF'v (38). The immunotoxin itself was obtained to more than 90% from the soluble fraction of°ter bacterial lysis, was monomeric and could be expressed and purified with a yield of approximately 0.5 mg per liter bacterial culture from simple shake flasks. These are excellent prospects for scale-up by high cell density fermentation (60). The high stability of the immunotoxin was confirmed by the large proportion of molecules that eluted in monomeric form after 20 h incubation at 37°C in PBS, a result that was also obtained by incubating the radioactive-labeled immunotoxin in serum. Addition of protease inhibitors prolonged the stability for more than 48 h, indicating that protein degradation was not due to intrinsic molecule instability, but rather was a side effect occurring during purification.
The addition of ETA and a second hexahistidine tag at the N-terminal end of the scFv did not interfere with the binding properties of this ligand.
In viva, the systemically administered 4DSMOCB-ETA was cleared from the blood with slightly slower kinetics when compared to the sc:Fv, probably as a consequence of its increased molecular size. The blood clearance rate inversely correlated with the amount of'radioactivity in the kidney which was lower for the immunotoxin than for the scFv (ID/g tissue 22.79% vs.
300%).
Although all doses of immunotoxin were well-tolerated and mice did not show any signs o.f illness such as weight loss, the accumulation of 4I)5M0C'B-ETA in liver, spleen and bone raised the issue of potential toxicity to these tissues. The inhibitory effect of ETA
on protein synthesis is known t<> induce severe hepatotoxicity by sensitizing hepatocytes to the action of TNF, which is released by Kupffer cells upon E'fA mediated T cell stimulation and induces liver cell necrosis (46,47 ). 'To assess the degree of liver damage upon 4DSMOCB-ETA treatment, immunocompetent mice received repeated doses of 5 pg (250 p,g.kg-I ) or 10 pg (500 pg.kg-1 ) immunotoxin every other day for three cycles. In both cases, the level of AC,TiAST activity in the plasma of~ treated mice did not change significantly compared to untreated controls. First signs of impaired liver function only appeared after treatment with a 20 pg ( 1 mg.kg-1 ) dose given twice every other day (7.5-fold increase over control). In line with these findings, histological analysis of liver specimens did not reveal any signs of E'TA-induced pathological changes, except for the two 20 pg treatments, which induced moderate hepatocyte necrosis. A recent report has shown that E'T'A-induced hepatotoxicity and VLS can be circumvented and enlarge the therapeutic window by pre-treatment with anti-inflammatory agents, such as indomethacin or soluble TNF
receptor and non-steroidal drugs, respectively (27,29). 'The lack of correlation between organ specific accumulation and toxicity of thc: tmmunotoxin strongly suggests that the radioactivity in these highly perfused organs simply reflects the presence o1'non-cell bound and noninternalized ~mmunotoxin in the large blood pool and capillary network. On the other hand, it is unclear tmmunotoxin 9 whether in specialized phagocytes such as Kupffer cells of the liver, internalized proteins <;an escape into the cytoplasm, e.g. to interact with the elongation factor-2, or are rapidly directed to lysosomal degradation (61). In addition, it remains to be determined whether hexahistidine tags can affect the biodistribution behaviour of recombinant proteins in viva. In the kidney the level of radioactivity is always higher with metal-labeled than with iodinated proteins which are prone to dehalogenation (6~,). Therefore, metal-labeled proteins probably more accurately reflect the real picture of in viva biodistribution (63).
(,onstruction and purification of 4DSM(.)(."B-ETA
To make the Ep-CAM-specific scFv 4DSMOCB into an immunotoxin, this scFv antibody was fused to a truncated ETA (ETAZsz_~,>H) by means of a ~0 amino acid long peptide linker (Figure 1 A). The C-terminal original ER retention sequence REDLK of wild-type ETA (aa 609-613) was replaced by the mammalian counterpart KDEL which increases the cytotoxic potency of the toxin m tumor cells (38, 45). Furthermore, we added a second hexahistidine sequence at the N-terminus of 4DSM()('B to increase the efficiency of purification by Ni2+-1DA affinity chromatography (Figure l A) The final construct encoded a protein of 64R amino acids with a theoretical isoelectric point of 5.9. figure lB show; a computer model of the mature 4DSM0C.'B-ETA
immunotoxin molecule.
During 1P'f(~ induction, more than 90% of the total immunotoxin detected by Western blot was found in the periplasmic soluble fraction of E. coli and was released upon cell fractionation. The Bnal product yield was 0.5 mg of a 95°/.. pure immunotoxin preparation per liter bacterial culture in standard shake t7asks. The product mtgrated at the expected size of approximately 70 kDa on SDS-PAGE (Figure 1C) and the theoretical M~ of 69,737 Da was verified by mass spectrometry.
I'he absence oi' proteolytic degradation was conftrmed by Western blot analysis (Figure l D).
lmmunoreactivity and stability of 4DSMOCB-ETA
Thermal stability and resistance to protease degradation of an nnmunotoxin is of paramount importance for its tumor targeting properties, and thus for therapeutic efficacy. To investigate the stability of 4DSMOC:B-E'TA, the fusion protein was incubated in PBS for different time periods at 37°(.' and the rate of degradation was analyzed by gel filtration essentially as described (38). As shown in Figure 2, upon a 4 h incubation at 37°C, 91°i~ of the immunotoxin molecules still eluted as monomers at the retention volume of 1.4 ml, corresponding to a M~ of approximately 66 kDa.
The amount of 4DSMOC B-ETA only slowly decreased with time and approximately 47% of the Immunotoxin 10 initial protein still eluted in monomeric form after 20 h at 37°C.
Similar results were obtained upon incubation of ''''"'Tc-labeled 4DSMOCB-ETA in human serum, further corroborating the suitability of the immunotoxin for in vivo application.
To assess the effect of the additional N-terminal hexahistidine tag on the antigen binding affinity., we determined the amount of immunoreactive immunotoxin in a binding assay as described (43).
Upon a 1 h incubation at 37°C, the y''"'TC-triearbonyl quantitatively bound to the histidine t;~gs of the immunotoxin. As determined in cell binding assays, g0-90% of the immunotoxin retained its binding activity for Ep-(."AM after the labeling procedure. The K" of the immunotoxin to Ep-C.'AM expressed on SW2 cells was determined to be 4 nM, which was essentially the same as observed for the scFv 4DSMOCB assessed in a similar test system (3H). The low level of immunotoxin degradation could he completely prevented by the addition of protease inhibitors even after a 48 h incubation at 37°C in PBS (data not shown). Thus, the immunotoxin 4DSM0<.'B-ETA retained all the favorable biophysical properties of the parental scFv.
Ep-CAM expression on tumor cell lines Ep-CAM is overexpressed in many solid tumors of diverse histological origins (5). As shown in Table t, the highest level of Ep-C'AM was expressed on HT29 cells (MFI 696.1 ), followed by MC'F7 (MFi 419.5), CAL27 (MF1 415.3) and SW2 cells (MFI 372.4). The cell tines RL and COLO320 cells did not express Ep-CAM and were used as antigen-negative controls.
Tumor cell line Tissue of origin Staining Controlb MFI"
Tumor cell line Tissue of' origin Staining Control' SW2 lung 372.4 t 4.0 3.4 ~ 0.5 HT29 colon 696.1 t 1 1.4 5.4 f 0.4 C AL27 tongue 415.3 t 4.8 5.6 t 0.3 MCF7 breast 419.5 ~ 1.1 4.5 t 0.6 RL B lymphoblast 4.2 t 0.2 4.0 t 0.2 COL0320 colon 5.5 t 0.1 5.1 t 0.1 " The values are expressed as mean fluorescence intensities (MFI t SD) of three independent FAC S analyses of tumor cell lines stained with the Ep-CAM-specific antibody KS 1 ~4.
~' lJnspecitic staining was assessed by incubation of cells with the FITC-conjugated secondary antibody alone.
Immunotoxin 1 1 C.'ytotoxicity of 4DSMOCB-ETA against tumor cells in vitro To determine the ability of 4DSMOCBI?TA to specifically inhibit the growth of Ep-CAM-positive tumor cells, MTT assays were performed. The immunotoxm was specifically cytotoxic against Ep-CAM-positive cell lines and did not affect the growth of'the Ep-(:AM-negative cells RL and C OLO320 in the broad range of concentrations tested (Figure 3). SW2, CAL2'7 and MCF7 cells were found to be equally sensitive to the cytotoxic effect of 4D5MOGB-ETA and their proliferation was inhibited with an IC50 of only 0.00 pM. Despite the highest level of Ep-CAM expression (Table 1 ), HT29 cells were found to be the least sensitive (IC50 of 0.2 pM). In the range of concentrations tested, the cyt.otoxicity of the immunotoxin was completely blocked by an excess of scFv 4DSMOC.B (data not shown)..
As determined in [3H]leucine incorporation assays (data not shown), treatment of SW2 cells with 4DSMOCB-ETA inhibited protein synthesis with an IC50 of 0.01 pM, and this effect showed the same doseresponse relationship as measured in the cell viability assays described above. Protein synthesis was not inhibited in the antigen-negative control cell line RL.
Tumor localization of 4DSMOCB-ETA in mice (n order to spare normal tissues from cytotoxic damage and employ the full cytotoxic potential of 4DSMO('B-ET'A demonstrated ire vitro for targeted cancer therapy, the selective and preferential localization of the immunotoxin to Ep-(."AM-positive tumors is a prerequisite.
We assessed the tumor localization properties of 4DSMOC'B-ETA in a biodistr~bution experiment in mice bearing established Ep-CAM-positive SW2 and Ep-C'AM-negative COLO320 xenografts at the contralateral flanks As shown in Table ?, the maximum dose of radiolabeled 4DSMOCB-E,TA
detected in SW2 tumors was 2.93'% ID/g after 4 h, which then gradually decreased to 1.95°~o ID/g after 24 h. After 48 h radioactivity was still 1.13°/> ID~g tumor tissue. In COL,O320 control tumors 4DSM0(~B-ETA localized with a maximum dose of 1.65% ID/g after 30 min, which then rapidly declined to 1.06% II)ig after 4 h and showed only background levels after 48 h. As expected from its larger size, 4DSMOCB-FT'A showed a slower blood clearance than the parental scFv 4DSMOCB (data shown in Table 2 for comparison).. After 24 h the total dose of 4DSM()CB-ETAin the blood was 0.42°r« ID~g, which was l.~-fold more than measured for the scFv (0.28% ID/g). Moreover, localization of the immunotoxin in SW2 tumors was also delayed compared to the scF'v, and the distribution of 4DSMOCB-ETA revealed a tumor:blood ratio of 5.38 after 48 h, which was comparable tc~ the ratio obtained with the scFv after 24 h.
Immunotoxin I 2 At each time point 4DSMOCB-ETA preferentially accumulated in Ep-CAM-positive tumors compared to COLO320 control tumor with a SW2:COL0320 ratio varying between 1.28 and 2.95. This indicates that. 4DSMOCB-ET'A was retained in Ep-CAM-positive tumors by specitic antibody-antigen interactions and cellular uptake, and suggests that its marginal accumulation in COLO320 control tumors may be do to the increase in vascular permeability often found in tumors. Analysis of normal tissues revealed that 4DSMOCB-ETA
localized also in the kidney, spleen, liver and to a lower extent in the bone tissue of the femur.
Biodistribution of f''~~"TcJ-labeled 41)SMOC'B-ETA in micc3 bearing SW2 and COL0320 tumor xenografts immunotoxin scFv ' 1l.) 30 min 1 h 4 h 16 h 24 h 48 h 24 h min (~~-;) (n=3) (n-:..3)(n=-3) (n3) (n:r-3) (n=3) (n=3) ~issw ~uID/g<'iolD/g %ID/g oID/g %IDlg ~olD/g %ID/g %ID/g Blood 27.21 17.46 10.08 2.23 f 0.57 0.42 0.21 f 0.28 f 4.26 t 3.28 t 2.89 0.:32 f (1.14 t 0.04 0.02 t 0.06 Heart 10.84 6.69 6.20 2.23 f 1.14 0.64 0.52 t 0.28 t 1.96 f 3.88 +. 1.10 0. I f 0.04 t 0.47 0 05 t 0.09 Lung t 1.56 8.38 6.21 2.59 f 1.25 0.97 0.77 t i..14 f 1.66 t ) r 0.71 0.26 f 0.13 ~ 0.12 0.22 t 0.60 Spleen 735 f 12.17 I 1.64 7.70 f 8.81 6.24 4.07 f 0.70 1.50 f 1.70 t 1.74 5.65 ~ 1.26 t 0.68 1.69 f 0.13 Kidney 22.49 32.68 33.50 42.54 32.98 22.79 16.54 300.(1 f: 8.28 f. 1.49 t 2.32 f 6.(11 .t 1.14 f 1.76 f 0.39 t 85 Stomach 0.92 1.2 3 2.72 0.85 r I .15 0.63 0.45 t 0.24 f ().30 f ().40 + I 0. I ~ ().69 ~ 0.09 0.09 f_ .3 3 3 0.24 Intestine 1.78 2. I 2.31 2.30 t 1.21 0.90 0.58 t 0.30 ~ (1.52 9 t i: 0.5'21.09 t f).22 t 0.09 0.06 ~ 0.07 (1.05 Liver I 5.47 20.44 I 9.97 20.2() 16.28 ( 3.70 8.44 f 2.38 t 3.24 f 0.70 t 3.77 f 1.26 t 2.51 f 1.83 0.49 t 0.52 Muscle 0.63 0.8() 0.57 0.58 ~ 0.37 0.26 0.16 f 0.10 t 1).14 ~ (1.15 i 0.07 0.19 ~ f1,()3~ 0.05 0.02 t 0.02 Bone (femur) 3.91 6.24 3.96 3.76 t 3.(15 2.85 I .08 0.06 f 0.97 t (1.96 .z 0.23 0.43 ~ ().37 t 0.49 ~ 0.20 t (').05 COL0320 tumor0.79 I .65 1.26 I .19 1.06 0.66 0 55 f ND
t 0.10 f (1.33 t: 0. t. 0.05 ~- 0.20 f 0.03 0.14 SW2 tumor 1.01 2.45 2.45 2.93 t 2.26 I .95 I 13 t 1.47 t 0.22 t 0.3:3 -r. 0.80 ~ 0.53 t 0.37 0.08 t 0.32 0.47 Ratios COL0321) tumor/blood(1.03 0. l0 0.13 0.48 2.09 1.57 2 61 ND
SW2 tumor/blood(1.04 0.14 0.24 1.31 3.97 4.(i4 5-38 5.25 SW2 tumor/COL.03201.28 1.48 1.94 2.76 1.9(l 2.95 2 05 ND
tumor " Biodistribution of9g"'Tc-labeled 4DSMOCB-ETA immunotoxin was determined following i.v injection in mice bearing SW2 and COL0320 tumor xenografts at contralateral site. Data represent the percentages of injected dose (ID t SD) per gram tissue. ND, not determined.
'' Ratios presented were calculated from averages of tumor: blood or tumoraumor ratios of Individual mice.
Taken from Willuda et al~ ( 1999) for comparison.
~mmunotoxin I :i Toxicity of 4DSMOCB-ETA in mice 'The unexpected high localization of radioactivity in liver., spleen and bone raised the issue of potential toxicity to these tissues under immunotoxin therapy. To assess the toxicity of escalating doses of 4DSMOCB-ETA, C57BL/6 mice were used as immunocompetent hosts which, in contrast to athymic mice, are more sensitive to wild-type E'TA-mediated liver damage (46,47).
Interestingly, the determination of ALT AST levels in the plasma of C57Bt./6 mice 24 h after treatment with three cycles of either 5 lig or i 0 pg immunotoxin given every other day, did not reveal immunotoxin mediated impairment of liver function (Figure 4). Elevated transaminase activity was only observed upon administration of two 20 pg doses of immunotoxin which equaled the activity measured in control mice upon administration of a toxic dose of wild-type ETA (46). In line with these results, few sites with necroriC hepatocytes were only found upon treatment with the highest immunotoxin dose. At all doses tested, analysis of the spleen and the cellular components of whole blood samples did not show any signs of histopathological changes or myelosuppression, respectively (data not shown). Thus, the accumulation of radioactivity in liver, spleen and bone does not reflect the amount of cell-bound or internalized immunotoxin in khese tissues Antitumor activity of 4DSMOCB-ETA
An essential requirement for the clinical use of anti-cancer agents is a significant antitumor activity demonstrated in animal models of human tumor xenografts. To investigate whether the potent in vitro cytotoxicity and the favorable tumor localization properties of 4DSMOCB-E:TA
translate into antitumor activity, mice bearing large established SW2, HT29 or CAL27 tumor xenogralts were treated by i.v. injection of the immunotoxin with two different dose schedules: (i) 45 pg total, with .5 p.g given every second day for B weeks, (ii) ~i0 pg total, with 10 lig given every second day for I week. Mice bearing Ep--CAM-negative ('O'L.O320 tumor xenogratts were used as controls. All treatment doses were well tolerated and mice did not show any signs of toxicity such as weight loss or impaired liver function (Figure 4).
As depicted in Figure 5, a significant inhibition of the growth of all Ep-CAM-positive tumors was achieved by treating mice with either the 5 p.g or the 10 wg dose schedule.
Treatment of mice bearing SW2 xenografts resulted m a shrinkage of the tumor volume to maximal 20% of' the initial size and a slight resumption of growth to a final 2.6-fold size increase at the end of the monitored period. A similar effect was achieved upon treatment of ('AL27 tumors, which were reduced to maximal 60% of the initial volume. Fifty days after start ot'treatment the median tmmunotoxin 14 tumor volume did not exceed 1.4-fold the initial size. 'Two mice out of 7 treated with the S pg dose showed complete tumor regression and remained tumor free. Neither CAL27 nor SW2 tumors showed a significant difference Gn their tumor response to the two treatment schedules.
For HT29 tumors strong growth inhibition was achieved with the 5 pg dose given for 3 weeks when sizes decreased to 0.7-fold of the initial volume. As already observed for CAL27 tumors, ~
out of 7 mice showed complete regression of their HT29 tumors. Unexpectedly, the efficacy of the 10 ltg schedule was comparatively luwer, indicating that for these tumors a long-term treatment is more effective. No antitumor effect ot'4DSMOCB-ETA was seen in mice bearing Ep-CAM-negative COLO320 control tumors (Figure 5).
Tumor cell lines The colorectal carcinoma cell lines HT29 (HTB-38), COL0320 (CL-220), the breast adenocarcinoma cell line MCF7 (HTB-22) and the non-Hodgkin"s lymphoma cell line RL (CRL,-2261 ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The squamc.~us cell carcinoma cell line' of the tongue CAL27 was kindly provided by Dr. S. D. Bernal., Dana-farber C.."ancer Institute, Boston, MA The small cell lung carcinoma cell line SW2 was raised in our laboratory. Except for C'Af.27, which was maintained in Dulbecco's Modified Eagle Medium (Life 'Technologies, Grand Island, NY), cell lines were grown in RPMI-1640 (Life Technologies Inc., Grand Island, NY). Both media were supplemented with 10%
FBS (Hyclone, Europe Ltd. ), 2 mM L-glutamine., 50 IU~ ml penicillin and 50 pg/ml streptomycin. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5°~~ C02.
Construction of the 4DSMOCB-ETA expression vector 'The sequence encoding a truncated form of ETA ('E'TA~s~.~,"H) was amplified by PCR from plasmid pSW200 (25) and cloned as an 1 164 by EcoRI-Hinc~III fragment downstream of the Ep-C.'AM-binding 4DSMOC'B scFv sequence present in the pIG6-based (39) 4I)SMOCB
scFv expression vector (38) The primers (Tox 1 ~
('TCGGAATT('GOTG(~(.'GCGGCGGAGTTCCCG
AAACCGTC'C: ACC'C.'CCJCC GGGTTCTTC: TGGT'TTA; Tox2: GTC'AAGCTTC.'TAC.'AGTTCGT
CTTTATGGTGATGCiTGGTGATCiCG(.'C"GGTT'TC('C'CK,GC"TG) introduced an EcoRI
lmmunotoxin ] 5 restriction site between scFv and toxin and a C-terminal hexahistidine tag followed by the endoplasmic reticulum ( ER) retention signal KDEL, a stop colon and a HindIII
restriction site.
To improve purity and yield during IMAC, a second hexahistidine tag was added at the N-terminus between the periplasmic signal sequence and the 4D5MOC'B coding region. To this end, two pairs of oligonucleotides (Xbal 5': ('TAGATAACG.AGGC~CAAAAAATGAAAAAGACAG
C'TA'TCGCGA'TTGCAGTGGCACTGGC'TGGTTTCGCTACCGT: Xbal 3': GCCACTGC'AAT
CGCGATAG("TGTCTTTTTCATTTT TTGCCCTCGT'fA'T; and EcoRV 5': AGCGCAG(~CCG
AC.CACCATC ATCACCATCA("CAT; ~;coRV 3': 'T("GTGATGGTGATGATGGTGGTCGGCC.
TGCGC".TACGGTAGCGAAACCAGCCAGT) were heated to 80°(', allowed to anneal by gradually cooling to room temperature and then ligated between the Xbal and EcoRV sites of pIG6-4DSM0('BETAH6KDEL. The sequence was experimentally confirmed.
F',XAMPLE 3 Expression and purification of 4D5MO('B-ETA
F'or periplasmic; expression of 4D5MOC'B-ETA the vector pIG6 was used, which places the gene under lac promoter control in SB536, an ~'. coli strain devoid of the periplasmic proteases HhoA
and HhoB (40). Five ml 2YT medium containing ampicillin (100 pgiml) were inoculated with a single bacterial colony containing the 41)SMOCB-E'TA expression plasmid and grown overnight at 25°C. The bacteria were diluted in one liter of 2YT' medium supplemented with 0.5% glucose and ampicillin ( 100 pg/ml) to reach an fi5s~, nm between 0~ 1 and 0.2 and transferred to 3-liter baffled shake flasks. The culture was further grown at 25°(' to an A~5"
nm of 0.5 and immunotoxin production was induced for 4 h by adding a final Concentration of 1 mM isopropyl-p-D-thiogalactopyranoside (IPTG, Sigma). The harvested pellet derived from a bacterial culture with a 6na1 ,A55~ nm of 6 was stored at -80°(' For purification, the pellet obtained from a one liter culture was resuspended in 25 ml lysis buffer, containing SO mM Tris-HC1 (pH
7.5), 300 mM
NaCI, 2 mM MgSO4 and supplemented with EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and DNase I. The bacterial suspension was lysed with two cycles m a French Pressure Cell press (SL.S Instruments, Urbana, IL), centrifuged at 48'000 g in a SS-:34 rotor for 30 min at 4°C" and subsequently filter-sterilized (0.22 pin). The immunotoxxn present in the cleared supernatant was pin°i~ed by chromatography using a BIOCAD-System (Perceptive BioSystems) with a NiZ+-iminodiacetic (IDA) column and a HQ/M-anion-exch;~nge column coupled in-line as described (41 ). Before the lysate was loaded, the Niz+-IDA column was equilibrated with 20 mM Tras (pH 7.5), 300 mM NaC:I_ After loading, the column was washed three times with different salt solutions, all buffered with 20 mM Tris (pH 7.5), in the lmmunotoxin t 6 order 300 mM, 510 mM and 90 mM NaC'1. Subsequently, the column was washed with 20 mM
Tris (pH 7.5), l0 mM imidazole, 90 mM NaCI, before the bound immunotoxin was eluted with the same solution containing 200 mM imidazole (pH 7.5). 'The eluate was directly loaded onto the HQ/M-anion-exchange column and the bound immunotoxin was eluted with a salt gradient of 90a 1000 mM NaCI, buffered with 20 mM Tris (pH 7.5). The fractions containing were collected and concentrated using a 10 kDa cutoff filter by centrifugation at 2000 g and 4°C
(Ultrafree-MC low protein binding, Millipore). The quality of purified 4DSMOCB-ETA was analyzed by a l0% SDS-polyacrylamide gel and Western blotting using a horseradish peroxidase (HRP)-conjugated anti-tetrahistidine antibody (QIAGEN, Elilden, Germany) diluted 1:5000 according to the manufacturer's recommendations.
Analytical gel tiltration and determination of thermal stability Ten micrograms of purified 4DSMOCB-EVTA were diluted in 50 pl PBS pH 7.4 containing 0.005°~o T'ween-20 and subsequently incubated at 37°C. Samples were analyzed at different time points (after 0 h, 2 h, 4 h, 8 h, 10 h and 20 h) by gel Eltration using the Smart system (Pharmaczaj Uppsala) with a Superose-12 PC3.2!30 column. The column was calibrated in the same buffer with three protein standards: alcohol dehydrogenase (M~ 150,000), bovine serum albumin ( M, 66,000) and carbonic anhydrase (M,-29,000). 'The same analytical setting was used to assess the thermal stability of the '''''"Tc-labeled immunotoxin after a 20 h incubation at 37°C in human serum. The amount of immunotoxin monomers was determined by ~-scintillation counting of the eluted fractions.
EXAMPL.,E S
Radiolabeling and determination of antigen binding affinity 4DSMOCB-ETA was radioactively labeled by stable site-specific coordination of ''~"'Tc-tricarbonyl trihydrate to the hexahistidine tags present in the protein sequence (42); This spontaneous reaction was induced by mixing 30 pl of immunotoxin solution ( 1 mg/ml) with one third volume of 1 M 2-[N-morpholinojethanesulfomc acid (MES) pH 6.H and one third volume of freshly synthesized ''''"'Tc-tricarbonyl compound. The mixture was incubated for 1 h at 37°C and the reaction was stopped by desalting over a Biospin-6 column (BioRad, Hercules, CA) equilibrated with PBS containing O.OOS°,~~~ Tween-20q according to the manufacturer's Immunotoxm I 7 recommendation. The percentage of immunoreactive immunotoxin was assessed as described by Lindmo and co-workers (43). The binding affinity of the ''y"'Tc-labeled immunotoxin was determined on SW2 cells in a radio-immunoassay (R1A), essentially as described for the scFv 4DSMOC'B (3H).
Cell growth assay Inhibition of cell growth upon treatment with 4DSMOCB-ETA was determined in standard MTT
assays based on the reduction of tetrazolium salt to formazan by the enzymes from viable cells (44). Briefly, 5,000 tumor cells were seeded in 9fi-well ELISA microplates in a total volume of 50 gl culture medium per well Immunotoxin concentrations ranging from 0.0001-100 pM were added m a total volume of 100 pl per well and cells were incubated fc7r 72 h under standard cell culture conditions. Ten microliters of a 10 mg%ml MTT (Fluka) solution were added to each well and the plates were incubated for further 90 min at 37°C. Cell lysis and formazan solubilization were achieved by addition of l00 pl lysis butter containing 20% SDS in 50%
dimethylformamide (pH 4.7 adjusted with a solution consisting of 80% acetate, 20% I M HCl) and the released formazan crystals were allowed to dissolve overnight at 37°C.
Absorption was quantified at 590 nm using a SPE.,(.'TRAmax 340 rnicroplate reader (Molecular Devices, Sunnyvale, CA). To demonstrate that the cytotoxicity of4D5MOCK-E'fA was due to inhibition of protein synthesis in oells, [3H]leuc~ne incorporation assays were performed as described ( 18).
Briefly, 2 x 104 <:ells per well in leucine-free cell culture medmm were seeded into 96-well plates and incubated with increasing concentrations of 4DSMOC.B-ETA diluted in leucine-free medium to a final volume of 200 pl. Cells incubated in leucine-free medium without immunotoxin were used as control. Upon a 24 h incubation at 37°(. under standard cell culture conditions, cells were pulsed with 10 p.l medium containing 1 yCi [4,5-;3HJleucine (specific radioactivity 5 TBq/mmol) per well for 6 h and harvested onto glass-fiber filters using a Harvester 96 (Tomtec, Hamden, CT). The radioactivity incorporated into cells was quantified m a Trilux 1450 Icquid scintillation MicroBeta counter (PerkinElmer Life Sciences, Wellesley, MA) and expressed as percentages relative to untreated controls.
~mmunotoxin I H
Flow cytometry Cell surface expression of Ep-CAM was quantified by flow cytometry using the mouse IgC~~a KS114 (BD PharMingen, San Diego, ('A). As secondary antibody a FITC-conjugated goat anti-mouse F(ab')2 IgG (H+L) (Zymed Laboratories, San Francisco, CA) was used. All staining steps were performed in a staining buffer consisting of PBS supplemented with 1 %
(w/v) BSA and 0.04% (w/v) sodium azide. Cells (5 x 105) were harvested, washed twice with ice-cold staining buffer and incubated on ice for 45 min ire a total volume of l00 lrl staining buffer containing l Irg of the first antibody. Cells were washed and further incubated with 400 ng of FITC-labeled antibody in a final volume of 100 pl. After 30 min on ice, cells were washed and resuspended in 300 p.l staining buffer for analysis. Fluorescence intensity was measured at 430 nm using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) and quantified using the CellQuestPro software (Becton Dickinsc7n, San Jose, CA ).
Mice Six to R weeks old female CD-1 (ICR nu/nu) mice (Charles River Laboratories, Sulzfeld, Germany) were used. They were kept under specific pathogen-free conditions according to the guidelines of the Veterinary Office of'the Kanton Zurich. Tumors were raised at the lateral flank by s.c. injection of 107 cells and randomized to constitute groups with an average tumor si~:e of l60 mm3 Ten weeks old female (.'S7Bl.,i6 mice (Janvier, Saint Isle. France) were used to determine immunotoxin-specific toxicity in immunocompetent animals, which are more sensitive to the f?TA-mediated T cell stimulation that results in the production of TNF
by Kupffer cells and perform by cytotoxic T cells.
Biodistribution study of 4DSMOC'B-ETA
To investigate the distribution of 4DSMO CB-ETA m mice, 6 wg yy'"Te-labeled (specific radioactivity of 98.y TBq/mmo() were diluted in a total volume of 150 pl PBS and were injected r.v.. mto mice bearing established SW2 and ("010320 tumor xenografts at the contralateral lmmunotoxin 19 flanks. Mice were sacrificed at different time points ( 10 min, 30 mm, 1 h, 4h, 16h, 24h and 48 h) after treatment and organs were removed to measure the accumulated radioactivity using a p-counter. 'The amount of radioactivity per gram organ was given as percentage of the total injected dose which was arbitrarily set to l00%, Cn viva toxicity of 4DSMOC'B-ET'A
Toxicity of the immunotoxin was determined in C'S7BL/6 mice by measuring ALT/AST acaivity in plasma upon repeated injections of escalating doses of 4D5MOC'B-ETA given every other day for three cycles (S Itg and 10 pg dose) or for two cycles (20 p,g dose). Whole blood samples were taken to assess the degree of myelosuppression based on alterations of' cellular components. In addition, tissue specimens from the livers and spleens of immunotoxin treated mice were analyzed for hcstopathological changes upon hematoxylin/eosin staining.
Antitumor activity of 4DSMOCB-ETA
Mice bearing tumor xenografts derived from the Ep-("AM-positive cell lines C'AL27, HT29 and SW2, and the Ep-C'AM-negative cell line COL0320 were treated i.v. every second day with either S ltg 4DSM()CB-ETA for a total of 9 applications (total dose 4S ltg), or with l0 lug every second day for a total of a applications (total dose 30 pg) in a volume of l00 pl PBS. Tumor xenografts from untreated mice were used as control Tumor size was calculated by measurement of the shortest and longest perpendicular diameter using digital calipers according to the formula (short diameter)' x (long diameter) x 0.s, lmmunotoxin REFERENCES
1. Gottesman, M. M. and Pastan, I. Biochemistry of multidrug resistance mediated by the multidrug transporter Annu. Rev. Biochem., 62: 385-427, 1993.
The present invention relates to the field of cancer therapy and particularly to targeted therapy using immunotoxms.
BACKGROUND OF THE INVENTION
The lack of significant advances in the treatment of metastatic or refractory cancers has stimulated the design of novel approaches to targeted cancer therapy such as the use of antibody~~
based cancer therapeutics Despite favorable initial responses, most advanced solid tumors develop resistance to standard treatments and relapse as incurable metastatic diseases ( 1 ). Since increasing the dose of conventional anticancer agents results in unacceptable side effects, the design of novel therapies based on the use of tumor selective targeting ligands and effector domains employing different mechanisms of action is of great rmportance. Antibodres targeting tumor-associated antigens and equipped with intrinsic cytotoxic or rmmunostimulatory effector functions have shown promising antitumor activity in preclinical and clinical studies (2-4).
Because of their different mechanism of action and especially because of their built-in targeting function that conventional anti-cancer agents do not have, immunotoxins may add new options for the treatment of malignancies resistant to conventional treatments (26,28). ETA and its homologues irreversibly block protein synthesis in cells by ADP-ribosylating a posttranslationally modified histidine residue of elongation factor 2, called diphthamide, which ultimately triggers apoptosis (48). Although resistance of°cells to ET'A was described as a consequence of the mutation of the crucial histidine residue or loss of enzyme activity required for diphthamide synthesis (49,5(1), this is a rather uncommon event and has not been confirmed in other cell systems. Nevertheless, the loss of the tumor antigen used for uptake of the antibody-toxin tizsion protein by the tumor cell is another conceivable mechanism of resistance.
An immunotoxin is a chimeric protein in which a toxin moiety is chemically or genetically linked to a monoclonal or recombinant antibody Antibodies with speciticities for various tumor-associated antigens have been investigated as carriers of"toxins, and the majority of those that target solid tumors have employed the effector function ot'truncated ET'A
(.ETA) which lacks the cell binding domain I (aa 1-252) (20), F.T A irreversibly inhibits protein synthesis by ADP-ribosylation caf elongation factor 2 and therefore has to gain access to its intracellular target in tire Immunotoxin cytoplasm (20,21 ). Thus, the most promising antigens for immunotoxin therapy are those that are efficiently internalized into tumor cells upon antibody binding by receptor-mediated endocytosis (22-25 ).
A number of chemical and recombinant immunotoxins that employ either plant or bacterial toxins as eff'eetor domains and that target distinct cell surface antigens associated with tumor cells have been shown to be potent and selective anti-cancer agents in preclinical studies (28). However, only few of them proved to be promising candidates for clinical use.
Several immunotoxins, either as chemically~linked first generation or recombinant second generation formats, have been tested in animal models and in patients with advanced solid tumors (20,26). Although early clinical data using imrnunotoxrn therapy for refractory tumors remains promising, the induction of neutralizing antibodies and dose-limiting side effects associated with VL,S or liver toxicity still remain obstacles to effective therapy (27-29). To overcome these limitations, more promising tumor-associated antigens have to be validated as targets for nc:w irnmunotoxin generations which are equipped with rationally engineered effector functions (2,30,31 ).
Mayor responses have been reported so far only for leukemias. In phase I
studies using LMI3-2, an FTA based scFv immunotoxin which targets lL-2 receptor p-chain, responses have been observed treating hematologic malignancies (51,52). Moreover, two thirds of the patients with refractory hairy-cell leukemia involved in a phase I study showed complete remission after treatment with 81.22, a recombinant F.'fA-based dsFv immunotoxin specific for the CD22 surface antigen (53)0 Both immunotoxins showed minor or reversible toxic side effects and thus merited to be involved in phase I studies for which currently a 'larger number of patients are being recruited.
T wo recombinant F;TA-based dsfv immunotoxins are also currently being evaluated for the treatment of advanced solid tumors. The immunotoxin SSI(dsFv)-PE3R is directed against cells expressing mesothelin, a protein normally produced by mesothelial cells and expressed also on malignant mesotheliomas and ovarian carcinomas (54). The immunotoxin LMB-9 has been derived from the monoclonal antibody B3 that targets the LewisY antigen (55), which is also widely expressed in epithelial tissues, a fact that contributes to safety concerns. LMB-9 and SS I (dsfv)-PE3R are currently being tested in phase 1 clinical trials in patients with advanced solid tumors and first results are eagerly awaited. In a previous study with 3H patients suffering from advanced carcinomas the chemical conjugate of~monoclonal antibody B3 and ETA induced one complete and one partial response (S6). In these studies vascular leakage due to capillary lmmunotoxin 4 damage was found to be dose-limiting, and subserauent preclinical investigations revealed significant binding of the B3 antibody to LewisY expressed on endothelial cells (57).
In addition to HER-2/neu (25), Ep-CAM represents another more promising target for antibody-based therapy of solid tumors due to its abundant expression in many carcinomas and its limited distribution in normal epithelial tissues (5). Although, Ep-C.AM expression is not exclusively restricted to tumor cells, Riethmuller and co-workers found that application of the anti-Ep-CAM
monoclonal antibody 17-1 A in patients with resected colorectal carcinoma or minimal residual disease reduced the overall mortality by 32%, decreased the recurrence rate by 23% (1), and reduced the number of distant metastases (8).
Ep-CAM t is a 40 kDa transmembrane protein overexpressed in many solid tumors including carcinomas of the lung, breast, ovary, colorectum and squamous cell carcinoma of the head and neck (5). The limited expression of Ep-c"AM in normal epithelial tissues (5,6) makes this antigen an attractive target for cellular and antibody-based immunotherapy (7-9).
Recently, a transgenic mouse model mimicking the Ep-(:SAM expression pattern in humans further validated the suitability of this target for imrnunotherapy by showing no localization of the monoclonal antibody MfW'31 in Ep-CAM-positive ruormal tissues ( 10).
The role of Ep-('AM in carcinogenesis and malignant progression is still unclear, but there is increasing evidence that it modulates cell-cell interactions ( 1 1 ) and that its expression correlates with the rate of cell proliferation ( 12) In addition, a promoting role of Ep-CAM in tissue invasion and metastasis has been suggested ( 13), and a strong correlation between Ep-CAM expression and tumor progression has been found in patients with squamous cell carcinoma of the head and neck'. Ep-C.'AM-specific antibodies have been used in imaging studies to detect primary tumors and localize distant. metastases in patients mth SCL,C (14) and NS("LC (15), to trigger antitumor immune responses ( 16), and to deliver cytotoxic effector molecules to tumors in preclinical models ( 17,18) and in patients ( 19).
~ The abbreviations used are: Ep-C'AM, epithelial cell adhesion molecule;
S('LC, ,mall cell lung cancer; NSC'L.C, non small cell lung cancer; ETA, P.ceudvmona.v aemn,s;lnacu exotoxin A; VL..S, vascular lean syndrome; seFv, single chain antibody fragment: dsFv, disulfide-stabilized ,ingle-chain antibody fragment;
FBS, fetal bovine serum; IMA(', innnobilized ion-metal affinity chromatography.; PAGE, polyacryiamide gel electrophoresis; PBS, phosphate buffered saline; MT'f, i-14,5-dimethylthiazol-?-yl]-?,5-etiphenyltetrazolium bromide;
F1TC' fluorescein isothiocyanate, MFI, mean fluorescence intensity; E(.iF, epithelial growth factor AI.T, alanme aminotransferase; AST, aspartate aminotransferase; TNF, tumor necrosis factor D. Tschudi, unpubLshed observation.
lmmunotoxin Although in a previous study we reported the ability of the chemically conjugated immunotoxin lYIO('31-ET'A to eradicate small tumor xenografts in mice as well as its failure to delay the growth of larger tumors ( 18), we concluded that, due to its relatively large size of 200 kDa, the immunotoxin was unable to homogeneously distribute wrthin the tumor mass and thus could only affect an insufficiently small proportion of clonogenic tumor cells. Support for this hypothesis is promded by others who reported an inverse correlation between immunotoxin size and efficacy (32j. The tumor targeting and tissue distribution properties of ammunotoxins can be substantially improved by using small scFv as targeting ligands (22,2ni,25,3:3-37). We have recently described the enhanced tumor localization of scFv 4DSM0('.B, which was derived by grafting the hypervariable loops of monoclonal antibody MO('3 l onto the humanized framework of'the anti-HF:R-2ineu scl~'v 4DS and by additionally changing eight critical care residues to obtain a high molecule stability (3H). We have now teound that the development ofa fully recombinant Ep-t."AM-specific single-chain immunotoxin based on 4DSMOCB, can be employed to achieve favorable tumor localization and potent antitumor activity against carcinomas of diverse histologrcal ongins in vivo.
SUMMARY OF THE INVENTION
The present invention is predicated on the finding that 4DSMO CB-ETA exerts significant growth inhibition upon systemic administration to mice bearing large established tumor xenografts ( 160 mm;' from colorectal, small cell lung or squamous cell carcinoma of the head and neck. The present invention reports on two dose schedules that were well-tolerated and proved to be very effective in inhibiting tumor growth. The three-week treatments with a total dose of 45 trg eradicated a significant fraction of°the tumors, and some mrce remained tumor-free during the whole study. However, in contrast, after completion of the shorter one week treatment with a total dose of 30 lrg 4DSMOCB-ETA, HT29 tr.unors rapidly resumed their growth.
BRIEF DESCRIPTION OF THE DRAWINGS
Thz present invention is illustrated but not limited by the attached drawings of which:
Frgure l A is a schematic representation of the scFv-toxin fusion protein precursor, which includes the ompA signal sequence for periplasmic expression. 'The scFv antibody fragment ~4D~M()(~B is fused to the Pseudomonas exotoxin A (ET'A;52_~os) by the linker shown. The protern is tlanked by two hexahiscidine tags, the C.'-terminal of' which precedes the ER retention signal 1CDEL.
lmmunotoxin 6 Figure ( B is a three-dimensional model of the mature 4DSMOCB-ETA., The structure of the scFv (VL in red, VH in orange), of ET'A;52..~r,x (domain II in light-blue, domain Ib in green and domain III in violet) and of the linking peptide (green) are shown. Both hexahistidine tags are indicated in yellow.
Figure 1 (.' is a copy of a chromatogram. 'Total extract of SB536 bacterial culture samples before (-) and after (+) IPTG induction and 10 p,g of 4DSMOCB-ETA immunotoxin purified by Ni2+-IDA and anion exchange affinity chromatography columns coupled in series, were analyzed on 10% SDS-PAGE under reducing conditions Figure I D shows the immunotoxm proteins present in the same samples as visualized on a Western blot using a HRP-conjugated anti-tetrahistidine antibody. Markers are shown in lane M:
myosin ( M~ 20H,000), w-galactosidase (Mr 1 19,000), bovine serum albumin (M,.
94,000), ovalbumm (Mr 51,100), carbonic anhydrase (Mr 35,400) and soyabean trypsin inhibitor (M~
2H,so0) Figure 2 shows the chromatograms before (0 h) and after 2 h, 4 h, H h, 10 h and 20 h incubation were recc'~rded at 2H0 nm. The monomers eluted at approximately l.4 ml as verified by calibration with the molecular weight standards alcohol dehydrogenase (A, M~ I 50,000), bovine serum albumin (B, M, H6,000) and carbonic anhydrase (C', M,29,000). which eluted at 1.31 ml, 1.38 ml and 1.54 ml, respectively (retentiun volumes shown by arrows). The stability of 4D5MOCB-ETA was assessed by size exclusion chromatography. 'The immunotoxin was incubated at a concentration of 200 ugiml at 37"C: in PBS and samples were analysed by gel filtration at different time points for comparison.
Figure 3 is a graphic depiction of cell growth measured in MTT assays as described in the "Material anc~ Methods" section. Data represent mean values of at least six independent determinations each carried out in quadruplicates (overall SD<5'%). Four Ep-(:AM-positive tumor cell lines were incubated for 72 h with 4DSMO('B-ETA at concentrations ranging from 0.0001 to 100 pM.
Figure 4 is a graphic representation of impairment of liver function upon treatment with 4DSMOC'B-ETA. C'S7BL/6 mice were treated every other day with escalating doses of 4D5MOC'B-ETA Two groups received 5 pg (?50 pg.kg-l ) or 10 pg (500 pg.kg-1 ) doses for three cycles, while another group was treated twice with 20 yg ( 1000 ltg.kg-1 ).
Twenty-four hours after Immunotoxin the last challenge the activities of plasma transaminases were determined and compared to mice treated with PBS (0 pg.kg-1 ). The transaminase activities of mice treated with a single lethal dose of wild-type E'TA- (85 pg.kg-1), as described by Schumann et al. (47)., are also shown. Data are expressed as the mean t SD (n = 3).
Figure 5 is a graphic representation of tumor growth illustrating the antitumor effect of ~DSMOCB-ETA in mice. Athymic mice bearing large tumor xenograf'ts ( 160 mm; in average) derived from the Ep-CAM-positive cell lines HT29, SW2 and (.'AC.27 remained untreated ( ? -) or were treated by i.v. injections every second day with either nine doses of 5 pg 4DSMOCB-ETA each for three weeks (-; -), or with three doses of 10 tig each (-'? -).
In a control experiment, mice bearing Ep-CAM-negative (.'OLO320 xenografts were also treated with 4DSMOCB-E.TA
according to the dose schedules mentioned above. 'The tumor size is given relative to the initial median tumor size of 160 mm3 at the start of treatment and data represent the mean values pSD
of'the various groups (n - 7).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The immunotoxin 4DSMOC:B-ETA was developed by fusing the highly stable humanized scFv 4DSMO('B (38) to a truncated form of hTA comprising amino acids 2.52-608 and the C-terminal eukaryotic ER retention sequence KDEI.. The 4DSMOCB-ETA is extremely potent in the femtomolar range and potently inhibits the growth of°carcinoma cells of diverse histological origins in a highly antigen-specific manner as demonstrated by an increase in the cytotoxic potency by more than four orders of magnitude, compared to antigen-negative cells. Moreover, the antigen-specific action of 4DSMOCE~-ETA was further corroborated in competition assays using an excess of scFv 4D5M0('B. Similar to the findings reported from immunotoxins targeting other tumor-associated antigens including HER-2/neu and E(iF
receptor (25,59), the cytotoxic activity of 4D5M0(:B-f=TA did not correlate with the amount of target antigen expressed on the tumor cell surface. Thus, it is likely that other cell type-specific parameters, such as rate of internalization, intracellular trafficking and fate of the enzyme domain are further determinants of immunotoxin efficacy. In terms of its in vitro cytotoxicity 4DSMOCB-ETA is the most potent Ep-C'AM specific iinmunotoxin that has been reported in the literature and was 1000-fold more potent than the chemical immunotoxin conjugate MOC'31-E:TA ( 18).
~, prerequisite for the optimal binding of antibody-based therapeutics to target antigens expressed an the surface of tumor cells and for efficient tumor localization is protein stability under physiological conditions. The extremely potent cytotoxicity shown by 4D5MOCB-ETA may at lmmunotoxin least partly be due to the stability of the targeting scF'v (38). The immunotoxin itself was obtained to more than 90% from the soluble fraction of°ter bacterial lysis, was monomeric and could be expressed and purified with a yield of approximately 0.5 mg per liter bacterial culture from simple shake flasks. These are excellent prospects for scale-up by high cell density fermentation (60). The high stability of the immunotoxin was confirmed by the large proportion of molecules that eluted in monomeric form after 20 h incubation at 37°C in PBS, a result that was also obtained by incubating the radioactive-labeled immunotoxin in serum. Addition of protease inhibitors prolonged the stability for more than 48 h, indicating that protein degradation was not due to intrinsic molecule instability, but rather was a side effect occurring during purification.
The addition of ETA and a second hexahistidine tag at the N-terminal end of the scFv did not interfere with the binding properties of this ligand.
In viva, the systemically administered 4DSMOCB-ETA was cleared from the blood with slightly slower kinetics when compared to the sc:Fv, probably as a consequence of its increased molecular size. The blood clearance rate inversely correlated with the amount of'radioactivity in the kidney which was lower for the immunotoxin than for the scFv (ID/g tissue 22.79% vs.
300%).
Although all doses of immunotoxin were well-tolerated and mice did not show any signs o.f illness such as weight loss, the accumulation of 4I)5M0C'B-ETA in liver, spleen and bone raised the issue of potential toxicity to these tissues. The inhibitory effect of ETA
on protein synthesis is known t<> induce severe hepatotoxicity by sensitizing hepatocytes to the action of TNF, which is released by Kupffer cells upon E'fA mediated T cell stimulation and induces liver cell necrosis (46,47 ). 'To assess the degree of liver damage upon 4DSMOCB-ETA treatment, immunocompetent mice received repeated doses of 5 pg (250 p,g.kg-I ) or 10 pg (500 pg.kg-1 ) immunotoxin every other day for three cycles. In both cases, the level of AC,TiAST activity in the plasma of~ treated mice did not change significantly compared to untreated controls. First signs of impaired liver function only appeared after treatment with a 20 pg ( 1 mg.kg-1 ) dose given twice every other day (7.5-fold increase over control). In line with these findings, histological analysis of liver specimens did not reveal any signs of E'TA-induced pathological changes, except for the two 20 pg treatments, which induced moderate hepatocyte necrosis. A recent report has shown that E'T'A-induced hepatotoxicity and VLS can be circumvented and enlarge the therapeutic window by pre-treatment with anti-inflammatory agents, such as indomethacin or soluble TNF
receptor and non-steroidal drugs, respectively (27,29). 'The lack of correlation between organ specific accumulation and toxicity of thc: tmmunotoxin strongly suggests that the radioactivity in these highly perfused organs simply reflects the presence o1'non-cell bound and noninternalized ~mmunotoxin in the large blood pool and capillary network. On the other hand, it is unclear tmmunotoxin 9 whether in specialized phagocytes such as Kupffer cells of the liver, internalized proteins <;an escape into the cytoplasm, e.g. to interact with the elongation factor-2, or are rapidly directed to lysosomal degradation (61). In addition, it remains to be determined whether hexahistidine tags can affect the biodistribution behaviour of recombinant proteins in viva. In the kidney the level of radioactivity is always higher with metal-labeled than with iodinated proteins which are prone to dehalogenation (6~,). Therefore, metal-labeled proteins probably more accurately reflect the real picture of in viva biodistribution (63).
(,onstruction and purification of 4DSM(.)(."B-ETA
To make the Ep-CAM-specific scFv 4DSMOCB into an immunotoxin, this scFv antibody was fused to a truncated ETA (ETAZsz_~,>H) by means of a ~0 amino acid long peptide linker (Figure 1 A). The C-terminal original ER retention sequence REDLK of wild-type ETA (aa 609-613) was replaced by the mammalian counterpart KDEL which increases the cytotoxic potency of the toxin m tumor cells (38, 45). Furthermore, we added a second hexahistidine sequence at the N-terminus of 4DSM()('B to increase the efficiency of purification by Ni2+-1DA affinity chromatography (Figure l A) The final construct encoded a protein of 64R amino acids with a theoretical isoelectric point of 5.9. figure lB show; a computer model of the mature 4DSM0C.'B-ETA
immunotoxin molecule.
During 1P'f(~ induction, more than 90% of the total immunotoxin detected by Western blot was found in the periplasmic soluble fraction of E. coli and was released upon cell fractionation. The Bnal product yield was 0.5 mg of a 95°/.. pure immunotoxin preparation per liter bacterial culture in standard shake t7asks. The product mtgrated at the expected size of approximately 70 kDa on SDS-PAGE (Figure 1C) and the theoretical M~ of 69,737 Da was verified by mass spectrometry.
I'he absence oi' proteolytic degradation was conftrmed by Western blot analysis (Figure l D).
lmmunoreactivity and stability of 4DSMOCB-ETA
Thermal stability and resistance to protease degradation of an nnmunotoxin is of paramount importance for its tumor targeting properties, and thus for therapeutic efficacy. To investigate the stability of 4DSMOC:B-E'TA, the fusion protein was incubated in PBS for different time periods at 37°(.' and the rate of degradation was analyzed by gel filtration essentially as described (38). As shown in Figure 2, upon a 4 h incubation at 37°C, 91°i~ of the immunotoxin molecules still eluted as monomers at the retention volume of 1.4 ml, corresponding to a M~ of approximately 66 kDa.
The amount of 4DSMOC B-ETA only slowly decreased with time and approximately 47% of the Immunotoxin 10 initial protein still eluted in monomeric form after 20 h at 37°C.
Similar results were obtained upon incubation of ''''"'Tc-labeled 4DSMOCB-ETA in human serum, further corroborating the suitability of the immunotoxin for in vivo application.
To assess the effect of the additional N-terminal hexahistidine tag on the antigen binding affinity., we determined the amount of immunoreactive immunotoxin in a binding assay as described (43).
Upon a 1 h incubation at 37°C, the y''"'TC-triearbonyl quantitatively bound to the histidine t;~gs of the immunotoxin. As determined in cell binding assays, g0-90% of the immunotoxin retained its binding activity for Ep-(."AM after the labeling procedure. The K" of the immunotoxin to Ep-C.'AM expressed on SW2 cells was determined to be 4 nM, which was essentially the same as observed for the scFv 4DSMOCB assessed in a similar test system (3H). The low level of immunotoxin degradation could he completely prevented by the addition of protease inhibitors even after a 48 h incubation at 37°C in PBS (data not shown). Thus, the immunotoxin 4DSM0<.'B-ETA retained all the favorable biophysical properties of the parental scFv.
Ep-CAM expression on tumor cell lines Ep-CAM is overexpressed in many solid tumors of diverse histological origins (5). As shown in Table t, the highest level of Ep-C'AM was expressed on HT29 cells (MFI 696.1 ), followed by MC'F7 (MFi 419.5), CAL27 (MF1 415.3) and SW2 cells (MFI 372.4). The cell tines RL and COLO320 cells did not express Ep-CAM and were used as antigen-negative controls.
Tumor cell line Tissue of origin Staining Controlb MFI"
Tumor cell line Tissue of' origin Staining Control' SW2 lung 372.4 t 4.0 3.4 ~ 0.5 HT29 colon 696.1 t 1 1.4 5.4 f 0.4 C AL27 tongue 415.3 t 4.8 5.6 t 0.3 MCF7 breast 419.5 ~ 1.1 4.5 t 0.6 RL B lymphoblast 4.2 t 0.2 4.0 t 0.2 COL0320 colon 5.5 t 0.1 5.1 t 0.1 " The values are expressed as mean fluorescence intensities (MFI t SD) of three independent FAC S analyses of tumor cell lines stained with the Ep-CAM-specific antibody KS 1 ~4.
~' lJnspecitic staining was assessed by incubation of cells with the FITC-conjugated secondary antibody alone.
Immunotoxin 1 1 C.'ytotoxicity of 4DSMOCB-ETA against tumor cells in vitro To determine the ability of 4DSMOCBI?TA to specifically inhibit the growth of Ep-CAM-positive tumor cells, MTT assays were performed. The immunotoxm was specifically cytotoxic against Ep-CAM-positive cell lines and did not affect the growth of'the Ep-(:AM-negative cells RL and C OLO320 in the broad range of concentrations tested (Figure 3). SW2, CAL2'7 and MCF7 cells were found to be equally sensitive to the cytotoxic effect of 4D5MOGB-ETA and their proliferation was inhibited with an IC50 of only 0.00 pM. Despite the highest level of Ep-CAM expression (Table 1 ), HT29 cells were found to be the least sensitive (IC50 of 0.2 pM). In the range of concentrations tested, the cyt.otoxicity of the immunotoxin was completely blocked by an excess of scFv 4DSMOC.B (data not shown)..
As determined in [3H]leucine incorporation assays (data not shown), treatment of SW2 cells with 4DSMOCB-ETA inhibited protein synthesis with an IC50 of 0.01 pM, and this effect showed the same doseresponse relationship as measured in the cell viability assays described above. Protein synthesis was not inhibited in the antigen-negative control cell line RL.
Tumor localization of 4DSMOCB-ETA in mice (n order to spare normal tissues from cytotoxic damage and employ the full cytotoxic potential of 4DSMO('B-ET'A demonstrated ire vitro for targeted cancer therapy, the selective and preferential localization of the immunotoxin to Ep-(."AM-positive tumors is a prerequisite.
We assessed the tumor localization properties of 4DSMOC'B-ETA in a biodistr~bution experiment in mice bearing established Ep-CAM-positive SW2 and Ep-C'AM-negative COLO320 xenografts at the contralateral flanks As shown in Table ?, the maximum dose of radiolabeled 4DSMOCB-E,TA
detected in SW2 tumors was 2.93'% ID/g after 4 h, which then gradually decreased to 1.95°~o ID/g after 24 h. After 48 h radioactivity was still 1.13°/> ID~g tumor tissue. In COL,O320 control tumors 4DSM0(~B-ETA localized with a maximum dose of 1.65% ID/g after 30 min, which then rapidly declined to 1.06% II)ig after 4 h and showed only background levels after 48 h. As expected from its larger size, 4DSMOCB-FT'A showed a slower blood clearance than the parental scFv 4DSMOCB (data shown in Table 2 for comparison).. After 24 h the total dose of 4DSM()CB-ETAin the blood was 0.42°r« ID~g, which was l.~-fold more than measured for the scFv (0.28% ID/g). Moreover, localization of the immunotoxin in SW2 tumors was also delayed compared to the scF'v, and the distribution of 4DSMOCB-ETA revealed a tumor:blood ratio of 5.38 after 48 h, which was comparable tc~ the ratio obtained with the scFv after 24 h.
Immunotoxin I 2 At each time point 4DSMOCB-ETA preferentially accumulated in Ep-CAM-positive tumors compared to COLO320 control tumor with a SW2:COL0320 ratio varying between 1.28 and 2.95. This indicates that. 4DSMOCB-ET'A was retained in Ep-CAM-positive tumors by specitic antibody-antigen interactions and cellular uptake, and suggests that its marginal accumulation in COLO320 control tumors may be do to the increase in vascular permeability often found in tumors. Analysis of normal tissues revealed that 4DSMOCB-ETA
localized also in the kidney, spleen, liver and to a lower extent in the bone tissue of the femur.
Biodistribution of f''~~"TcJ-labeled 41)SMOC'B-ETA in micc3 bearing SW2 and COL0320 tumor xenografts immunotoxin scFv ' 1l.) 30 min 1 h 4 h 16 h 24 h 48 h 24 h min (~~-;) (n=3) (n-:..3)(n=-3) (n3) (n:r-3) (n=3) (n=3) ~issw ~uID/g<'iolD/g %ID/g oID/g %IDlg ~olD/g %ID/g %ID/g Blood 27.21 17.46 10.08 2.23 f 0.57 0.42 0.21 f 0.28 f 4.26 t 3.28 t 2.89 0.:32 f (1.14 t 0.04 0.02 t 0.06 Heart 10.84 6.69 6.20 2.23 f 1.14 0.64 0.52 t 0.28 t 1.96 f 3.88 +. 1.10 0. I f 0.04 t 0.47 0 05 t 0.09 Lung t 1.56 8.38 6.21 2.59 f 1.25 0.97 0.77 t i..14 f 1.66 t ) r 0.71 0.26 f 0.13 ~ 0.12 0.22 t 0.60 Spleen 735 f 12.17 I 1.64 7.70 f 8.81 6.24 4.07 f 0.70 1.50 f 1.70 t 1.74 5.65 ~ 1.26 t 0.68 1.69 f 0.13 Kidney 22.49 32.68 33.50 42.54 32.98 22.79 16.54 300.(1 f: 8.28 f. 1.49 t 2.32 f 6.(11 .t 1.14 f 1.76 f 0.39 t 85 Stomach 0.92 1.2 3 2.72 0.85 r I .15 0.63 0.45 t 0.24 f ().30 f ().40 + I 0. I ~ ().69 ~ 0.09 0.09 f_ .3 3 3 0.24 Intestine 1.78 2. I 2.31 2.30 t 1.21 0.90 0.58 t 0.30 ~ (1.52 9 t i: 0.5'21.09 t f).22 t 0.09 0.06 ~ 0.07 (1.05 Liver I 5.47 20.44 I 9.97 20.2() 16.28 ( 3.70 8.44 f 2.38 t 3.24 f 0.70 t 3.77 f 1.26 t 2.51 f 1.83 0.49 t 0.52 Muscle 0.63 0.8() 0.57 0.58 ~ 0.37 0.26 0.16 f 0.10 t 1).14 ~ (1.15 i 0.07 0.19 ~ f1,()3~ 0.05 0.02 t 0.02 Bone (femur) 3.91 6.24 3.96 3.76 t 3.(15 2.85 I .08 0.06 f 0.97 t (1.96 .z 0.23 0.43 ~ ().37 t 0.49 ~ 0.20 t (').05 COL0320 tumor0.79 I .65 1.26 I .19 1.06 0.66 0 55 f ND
t 0.10 f (1.33 t: 0. t. 0.05 ~- 0.20 f 0.03 0.14 SW2 tumor 1.01 2.45 2.45 2.93 t 2.26 I .95 I 13 t 1.47 t 0.22 t 0.3:3 -r. 0.80 ~ 0.53 t 0.37 0.08 t 0.32 0.47 Ratios COL0321) tumor/blood(1.03 0. l0 0.13 0.48 2.09 1.57 2 61 ND
SW2 tumor/blood(1.04 0.14 0.24 1.31 3.97 4.(i4 5-38 5.25 SW2 tumor/COL.03201.28 1.48 1.94 2.76 1.9(l 2.95 2 05 ND
tumor " Biodistribution of9g"'Tc-labeled 4DSMOCB-ETA immunotoxin was determined following i.v injection in mice bearing SW2 and COL0320 tumor xenografts at contralateral site. Data represent the percentages of injected dose (ID t SD) per gram tissue. ND, not determined.
'' Ratios presented were calculated from averages of tumor: blood or tumoraumor ratios of Individual mice.
Taken from Willuda et al~ ( 1999) for comparison.
~mmunotoxin I :i Toxicity of 4DSMOCB-ETA in mice 'The unexpected high localization of radioactivity in liver., spleen and bone raised the issue of potential toxicity to these tissues under immunotoxin therapy. To assess the toxicity of escalating doses of 4DSMOCB-ETA, C57BL/6 mice were used as immunocompetent hosts which, in contrast to athymic mice, are more sensitive to wild-type E'TA-mediated liver damage (46,47).
Interestingly, the determination of ALT AST levels in the plasma of C57Bt./6 mice 24 h after treatment with three cycles of either 5 lig or i 0 pg immunotoxin given every other day, did not reveal immunotoxin mediated impairment of liver function (Figure 4). Elevated transaminase activity was only observed upon administration of two 20 pg doses of immunotoxin which equaled the activity measured in control mice upon administration of a toxic dose of wild-type ETA (46). In line with these results, few sites with necroriC hepatocytes were only found upon treatment with the highest immunotoxin dose. At all doses tested, analysis of the spleen and the cellular components of whole blood samples did not show any signs of histopathological changes or myelosuppression, respectively (data not shown). Thus, the accumulation of radioactivity in liver, spleen and bone does not reflect the amount of cell-bound or internalized immunotoxin in khese tissues Antitumor activity of 4DSMOCB-ETA
An essential requirement for the clinical use of anti-cancer agents is a significant antitumor activity demonstrated in animal models of human tumor xenografts. To investigate whether the potent in vitro cytotoxicity and the favorable tumor localization properties of 4DSMOCB-E:TA
translate into antitumor activity, mice bearing large established SW2, HT29 or CAL27 tumor xenogralts were treated by i.v. injection of the immunotoxin with two different dose schedules: (i) 45 pg total, with .5 p.g given every second day for B weeks, (ii) ~i0 pg total, with 10 lig given every second day for I week. Mice bearing Ep--CAM-negative ('O'L.O320 tumor xenogratts were used as controls. All treatment doses were well tolerated and mice did not show any signs of toxicity such as weight loss or impaired liver function (Figure 4).
As depicted in Figure 5, a significant inhibition of the growth of all Ep-CAM-positive tumors was achieved by treating mice with either the 5 p.g or the 10 wg dose schedule.
Treatment of mice bearing SW2 xenografts resulted m a shrinkage of the tumor volume to maximal 20% of' the initial size and a slight resumption of growth to a final 2.6-fold size increase at the end of the monitored period. A similar effect was achieved upon treatment of ('AL27 tumors, which were reduced to maximal 60% of the initial volume. Fifty days after start ot'treatment the median tmmunotoxin 14 tumor volume did not exceed 1.4-fold the initial size. 'Two mice out of 7 treated with the S pg dose showed complete tumor regression and remained tumor free. Neither CAL27 nor SW2 tumors showed a significant difference Gn their tumor response to the two treatment schedules.
For HT29 tumors strong growth inhibition was achieved with the 5 pg dose given for 3 weeks when sizes decreased to 0.7-fold of the initial volume. As already observed for CAL27 tumors, ~
out of 7 mice showed complete regression of their HT29 tumors. Unexpectedly, the efficacy of the 10 ltg schedule was comparatively luwer, indicating that for these tumors a long-term treatment is more effective. No antitumor effect ot'4DSMOCB-ETA was seen in mice bearing Ep-CAM-negative COLO320 control tumors (Figure 5).
Tumor cell lines The colorectal carcinoma cell lines HT29 (HTB-38), COL0320 (CL-220), the breast adenocarcinoma cell line MCF7 (HTB-22) and the non-Hodgkin"s lymphoma cell line RL (CRL,-2261 ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The squamc.~us cell carcinoma cell line' of the tongue CAL27 was kindly provided by Dr. S. D. Bernal., Dana-farber C.."ancer Institute, Boston, MA The small cell lung carcinoma cell line SW2 was raised in our laboratory. Except for C'Af.27, which was maintained in Dulbecco's Modified Eagle Medium (Life 'Technologies, Grand Island, NY), cell lines were grown in RPMI-1640 (Life Technologies Inc., Grand Island, NY). Both media were supplemented with 10%
FBS (Hyclone, Europe Ltd. ), 2 mM L-glutamine., 50 IU~ ml penicillin and 50 pg/ml streptomycin. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5°~~ C02.
Construction of the 4DSMOCB-ETA expression vector 'The sequence encoding a truncated form of ETA ('E'TA~s~.~,"H) was amplified by PCR from plasmid pSW200 (25) and cloned as an 1 164 by EcoRI-Hinc~III fragment downstream of the Ep-C.'AM-binding 4DSMOC'B scFv sequence present in the pIG6-based (39) 4I)SMOCB
scFv expression vector (38) The primers (Tox 1 ~
('TCGGAATT('GOTG(~(.'GCGGCGGAGTTCCCG
AAACCGTC'C: ACC'C.'CCJCC GGGTTCTTC: TGGT'TTA; Tox2: GTC'AAGCTTC.'TAC.'AGTTCGT
CTTTATGGTGATGCiTGGTGATCiCG(.'C"GGTT'TC('C'CK,GC"TG) introduced an EcoRI
lmmunotoxin ] 5 restriction site between scFv and toxin and a C-terminal hexahistidine tag followed by the endoplasmic reticulum ( ER) retention signal KDEL, a stop colon and a HindIII
restriction site.
To improve purity and yield during IMAC, a second hexahistidine tag was added at the N-terminus between the periplasmic signal sequence and the 4D5MOC'B coding region. To this end, two pairs of oligonucleotides (Xbal 5': ('TAGATAACG.AGGC~CAAAAAATGAAAAAGACAG
C'TA'TCGCGA'TTGCAGTGGCACTGGC'TGGTTTCGCTACCGT: Xbal 3': GCCACTGC'AAT
CGCGATAG("TGTCTTTTTCATTTT TTGCCCTCGT'fA'T; and EcoRV 5': AGCGCAG(~CCG
AC.CACCATC ATCACCATCA("CAT; ~;coRV 3': 'T("GTGATGGTGATGATGGTGGTCGGCC.
TGCGC".TACGGTAGCGAAACCAGCCAGT) were heated to 80°(', allowed to anneal by gradually cooling to room temperature and then ligated between the Xbal and EcoRV sites of pIG6-4DSM0('BETAH6KDEL. The sequence was experimentally confirmed.
F',XAMPLE 3 Expression and purification of 4D5MO('B-ETA
F'or periplasmic; expression of 4D5MOC'B-ETA the vector pIG6 was used, which places the gene under lac promoter control in SB536, an ~'. coli strain devoid of the periplasmic proteases HhoA
and HhoB (40). Five ml 2YT medium containing ampicillin (100 pgiml) were inoculated with a single bacterial colony containing the 41)SMOCB-E'TA expression plasmid and grown overnight at 25°C. The bacteria were diluted in one liter of 2YT' medium supplemented with 0.5% glucose and ampicillin ( 100 pg/ml) to reach an fi5s~, nm between 0~ 1 and 0.2 and transferred to 3-liter baffled shake flasks. The culture was further grown at 25°(' to an A~5"
nm of 0.5 and immunotoxin production was induced for 4 h by adding a final Concentration of 1 mM isopropyl-p-D-thiogalactopyranoside (IPTG, Sigma). The harvested pellet derived from a bacterial culture with a 6na1 ,A55~ nm of 6 was stored at -80°(' For purification, the pellet obtained from a one liter culture was resuspended in 25 ml lysis buffer, containing SO mM Tris-HC1 (pH
7.5), 300 mM
NaCI, 2 mM MgSO4 and supplemented with EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and DNase I. The bacterial suspension was lysed with two cycles m a French Pressure Cell press (SL.S Instruments, Urbana, IL), centrifuged at 48'000 g in a SS-:34 rotor for 30 min at 4°C" and subsequently filter-sterilized (0.22 pin). The immunotoxxn present in the cleared supernatant was pin°i~ed by chromatography using a BIOCAD-System (Perceptive BioSystems) with a NiZ+-iminodiacetic (IDA) column and a HQ/M-anion-exch;~nge column coupled in-line as described (41 ). Before the lysate was loaded, the Niz+-IDA column was equilibrated with 20 mM Tras (pH 7.5), 300 mM NaC:I_ After loading, the column was washed three times with different salt solutions, all buffered with 20 mM Tris (pH 7.5), in the lmmunotoxin t 6 order 300 mM, 510 mM and 90 mM NaC'1. Subsequently, the column was washed with 20 mM
Tris (pH 7.5), l0 mM imidazole, 90 mM NaCI, before the bound immunotoxin was eluted with the same solution containing 200 mM imidazole (pH 7.5). 'The eluate was directly loaded onto the HQ/M-anion-exchange column and the bound immunotoxin was eluted with a salt gradient of 90a 1000 mM NaCI, buffered with 20 mM Tris (pH 7.5). The fractions containing were collected and concentrated using a 10 kDa cutoff filter by centrifugation at 2000 g and 4°C
(Ultrafree-MC low protein binding, Millipore). The quality of purified 4DSMOCB-ETA was analyzed by a l0% SDS-polyacrylamide gel and Western blotting using a horseradish peroxidase (HRP)-conjugated anti-tetrahistidine antibody (QIAGEN, Elilden, Germany) diluted 1:5000 according to the manufacturer's recommendations.
Analytical gel tiltration and determination of thermal stability Ten micrograms of purified 4DSMOCB-EVTA were diluted in 50 pl PBS pH 7.4 containing 0.005°~o T'ween-20 and subsequently incubated at 37°C. Samples were analyzed at different time points (after 0 h, 2 h, 4 h, 8 h, 10 h and 20 h) by gel Eltration using the Smart system (Pharmaczaj Uppsala) with a Superose-12 PC3.2!30 column. The column was calibrated in the same buffer with three protein standards: alcohol dehydrogenase (M~ 150,000), bovine serum albumin ( M, 66,000) and carbonic anhydrase (M,-29,000). 'The same analytical setting was used to assess the thermal stability of the '''''"Tc-labeled immunotoxin after a 20 h incubation at 37°C in human serum. The amount of immunotoxin monomers was determined by ~-scintillation counting of the eluted fractions.
EXAMPL.,E S
Radiolabeling and determination of antigen binding affinity 4DSMOCB-ETA was radioactively labeled by stable site-specific coordination of ''~"'Tc-tricarbonyl trihydrate to the hexahistidine tags present in the protein sequence (42); This spontaneous reaction was induced by mixing 30 pl of immunotoxin solution ( 1 mg/ml) with one third volume of 1 M 2-[N-morpholinojethanesulfomc acid (MES) pH 6.H and one third volume of freshly synthesized ''''"'Tc-tricarbonyl compound. The mixture was incubated for 1 h at 37°C and the reaction was stopped by desalting over a Biospin-6 column (BioRad, Hercules, CA) equilibrated with PBS containing O.OOS°,~~~ Tween-20q according to the manufacturer's Immunotoxm I 7 recommendation. The percentage of immunoreactive immunotoxin was assessed as described by Lindmo and co-workers (43). The binding affinity of the ''y"'Tc-labeled immunotoxin was determined on SW2 cells in a radio-immunoassay (R1A), essentially as described for the scFv 4DSMOC'B (3H).
Cell growth assay Inhibition of cell growth upon treatment with 4DSMOCB-ETA was determined in standard MTT
assays based on the reduction of tetrazolium salt to formazan by the enzymes from viable cells (44). Briefly, 5,000 tumor cells were seeded in 9fi-well ELISA microplates in a total volume of 50 gl culture medium per well Immunotoxin concentrations ranging from 0.0001-100 pM were added m a total volume of 100 pl per well and cells were incubated fc7r 72 h under standard cell culture conditions. Ten microliters of a 10 mg%ml MTT (Fluka) solution were added to each well and the plates were incubated for further 90 min at 37°C. Cell lysis and formazan solubilization were achieved by addition of l00 pl lysis butter containing 20% SDS in 50%
dimethylformamide (pH 4.7 adjusted with a solution consisting of 80% acetate, 20% I M HCl) and the released formazan crystals were allowed to dissolve overnight at 37°C.
Absorption was quantified at 590 nm using a SPE.,(.'TRAmax 340 rnicroplate reader (Molecular Devices, Sunnyvale, CA). To demonstrate that the cytotoxicity of4D5MOCK-E'fA was due to inhibition of protein synthesis in oells, [3H]leuc~ne incorporation assays were performed as described ( 18).
Briefly, 2 x 104 <:ells per well in leucine-free cell culture medmm were seeded into 96-well plates and incubated with increasing concentrations of 4DSMOC.B-ETA diluted in leucine-free medium to a final volume of 200 pl. Cells incubated in leucine-free medium without immunotoxin were used as control. Upon a 24 h incubation at 37°(. under standard cell culture conditions, cells were pulsed with 10 p.l medium containing 1 yCi [4,5-;3HJleucine (specific radioactivity 5 TBq/mmol) per well for 6 h and harvested onto glass-fiber filters using a Harvester 96 (Tomtec, Hamden, CT). The radioactivity incorporated into cells was quantified m a Trilux 1450 Icquid scintillation MicroBeta counter (PerkinElmer Life Sciences, Wellesley, MA) and expressed as percentages relative to untreated controls.
~mmunotoxin I H
Flow cytometry Cell surface expression of Ep-CAM was quantified by flow cytometry using the mouse IgC~~a KS114 (BD PharMingen, San Diego, ('A). As secondary antibody a FITC-conjugated goat anti-mouse F(ab')2 IgG (H+L) (Zymed Laboratories, San Francisco, CA) was used. All staining steps were performed in a staining buffer consisting of PBS supplemented with 1 %
(w/v) BSA and 0.04% (w/v) sodium azide. Cells (5 x 105) were harvested, washed twice with ice-cold staining buffer and incubated on ice for 45 min ire a total volume of l00 lrl staining buffer containing l Irg of the first antibody. Cells were washed and further incubated with 400 ng of FITC-labeled antibody in a final volume of 100 pl. After 30 min on ice, cells were washed and resuspended in 300 p.l staining buffer for analysis. Fluorescence intensity was measured at 430 nm using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) and quantified using the CellQuestPro software (Becton Dickinsc7n, San Jose, CA ).
Mice Six to R weeks old female CD-1 (ICR nu/nu) mice (Charles River Laboratories, Sulzfeld, Germany) were used. They were kept under specific pathogen-free conditions according to the guidelines of the Veterinary Office of'the Kanton Zurich. Tumors were raised at the lateral flank by s.c. injection of 107 cells and randomized to constitute groups with an average tumor si~:e of l60 mm3 Ten weeks old female (.'S7Bl.,i6 mice (Janvier, Saint Isle. France) were used to determine immunotoxin-specific toxicity in immunocompetent animals, which are more sensitive to the f?TA-mediated T cell stimulation that results in the production of TNF
by Kupffer cells and perform by cytotoxic T cells.
Biodistribution study of 4DSMOC'B-ETA
To investigate the distribution of 4DSMO CB-ETA m mice, 6 wg yy'"Te-labeled (specific radioactivity of 98.y TBq/mmo() were diluted in a total volume of 150 pl PBS and were injected r.v.. mto mice bearing established SW2 and ("010320 tumor xenografts at the contralateral lmmunotoxin 19 flanks. Mice were sacrificed at different time points ( 10 min, 30 mm, 1 h, 4h, 16h, 24h and 48 h) after treatment and organs were removed to measure the accumulated radioactivity using a p-counter. 'The amount of radioactivity per gram organ was given as percentage of the total injected dose which was arbitrarily set to l00%, Cn viva toxicity of 4DSMOC'B-ET'A
Toxicity of the immunotoxin was determined in C'S7BL/6 mice by measuring ALT/AST acaivity in plasma upon repeated injections of escalating doses of 4D5MOC'B-ETA given every other day for three cycles (S Itg and 10 pg dose) or for two cycles (20 p,g dose). Whole blood samples were taken to assess the degree of myelosuppression based on alterations of' cellular components. In addition, tissue specimens from the livers and spleens of immunotoxin treated mice were analyzed for hcstopathological changes upon hematoxylin/eosin staining.
Antitumor activity of 4DSMOCB-ETA
Mice bearing tumor xenografts derived from the Ep-("AM-positive cell lines C'AL27, HT29 and SW2, and the Ep-C'AM-negative cell line COL0320 were treated i.v. every second day with either S ltg 4DSM()CB-ETA for a total of 9 applications (total dose 4S ltg), or with l0 lug every second day for a total of a applications (total dose 30 pg) in a volume of l00 pl PBS. Tumor xenografts from untreated mice were used as control Tumor size was calculated by measurement of the shortest and longest perpendicular diameter using digital calipers according to the formula (short diameter)' x (long diameter) x 0.s, lmmunotoxin REFERENCES
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Claims (8)
1. A method of treating a mammal, including a human, in need of such treatment with an immunotoxin, said immunotoxin comprising an antigen binding polypeptide; which specifically recognizes Ep-CAM conjugated to a toxin selected from the group comprised of gelonin, bouganin, saporin, pseudomonas endotoxin (PE), ricin A chain, bryodin, diphtheria toxin or restrictocin, said method comprising administering the immunotoxin at least every 2nd day, for at least 12 days, preferably l4-28 days, in an amount of at least 5 pg per kg per dose, preferably at least approximately 7.5 pg per kg per dose.
2. A method according to claim 1, wherein at least 7, 8, 9 or 10 doses are administered in an amount of 7.5 pg per kg.
3. A method according to claim 1 or 2, wherein said toxin is a ribosome inactivating protein.
4. A method according to claim 3, wherein said ribosome inactivating protein is PE.
5. A method according to claim 1, 2, 3 or 4, wherein said antigen binding polypeptide is preferably selected from the group comprising Fab, Fab, scFv, single domain antibody fragments, disulfide stabilized Fvs, and dimers of the foregoing.
6. A method according to claim 5, wherein said antigen binding polypeptide is a scFv.
7 A method according to any of the preceding claims, wherein said antigen binding polypeptide competitively inhibits 4D5MOCB-ETA from binding to Ep-CAM.
8. The use of an immunotoxin comprising an antigen binding polypeptide which specifically binds to Ep-CAM for preparing an immunotoxin for treatment of cancer according to the method defined in claim 1.
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US20150018526A1 (en) * | 2004-03-19 | 2015-01-15 | Merck Patent Gmbh | Cytotoxins comprising modified bouganin toxin for the treatment of cancer |
US9822182B2 (en) | 2013-10-02 | 2017-11-21 | Viventia Bio Inc. | Anti-EPCAM antibodies and methods of use |
US10576163B2 (en) | 2015-03-12 | 2020-03-03 | Viventia Bio Inc. | Methods of treatment for EpCAM positive bladder cancer |
US10583198B2 (en) | 2015-03-12 | 2020-03-10 | Viventia Bio Inc. | Dosing strategies for targeting EPCAM positive bladder cancer |
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US10858429B2 (en) | 2003-04-30 | 2020-12-08 | University Of Zurich | Methods for treating cancer using an immunotoxin |
US20150018526A1 (en) * | 2004-03-19 | 2015-01-15 | Merck Patent Gmbh | Cytotoxins comprising modified bouganin toxin for the treatment of cancer |
WO2014166002A1 (en) * | 2013-04-12 | 2014-10-16 | Viventia Bio Inc. | Compositions and methods for detection and treatment of hepatocellular carcinoma |
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