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[Cancer Research 61, 8737-8742, December 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Human Angiogenin Fused to Human CD30 Ligand (Ang-CD30L) Exhibits Specific Cytotoxicity against CD30-positive Lymphoma1

Michael Huhn, Stephanie Sasse, Mehmet K. Tur, Bärbel Matthey, Timo Schinköthe, Susanna M. Rybak, Stefan Barth2 and Andreas Engert

Fraunhofer IME, Department of Pharmaceutical Product Development, 52074 Aachen, Germany [M. K. T., M. H., S. B.]; Laboratory of Immunotherapy, Department I of Internal Medicine, University Hospital Cologne, 50931 Cologne, Germany [S. S., B. M., T. S., A. E.]; Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702 [S. M. R.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl ß-D-thiogalactoside induction, the Mr 37,000 His10-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His10-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30+ Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC50 = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hodgkin’s lymphoma is one of the best suited malignancies for targeted immunotherapy for several reasons: (a) the lymphocyte activation marker CD30 is expressed in high copy numbers on H-RS3 cells; (b) the number of malignant cells that needs to be killed is small because the majority of cells in Hodgkin’s lymphoma are nonmalignant reactive cells; (c) Hodgkin tumors are usually well vascularized, suggesting sufficient access of an immunotherapeutic agent like an IT to the target cells; and (d) although Hodgkin’s lymphoma is known to respond well to chemotherapy, residual tumor cells remaining after first-line treatment have been demonstrated to correlate with the probability of a later relapse. Because the selective elimination of residual H-RS cells might enhance the number of patients being cured, it seems feasible to eradicate bulky disease by conventional therapy first and then to administer ITs to kill residual H-RS cells. One of the most promising target antigens for immunotherapy of malignant lymphoma such as Hodgkin’s lymphoma or anaplastic large cell lymphoma is the CD30 receptor. This antigen was originally discovered on cultured H-RS cells using the moab Ki-1 (1) . The gene encoding the CD30 receptor molecule (2) is located on chromosome 1p36. The naturally occurring CD30 ligand has also been identified and cloned (3) . The CD30/CD30 ligand system triggers cytolytic cell death in malignant lymphoma cell lines and induces proliferation and cytokine production in T cells or neutrophils (4) . Moabs against CD30 have been explored as vehicles for cytostatic drugs (5) or plant toxins (6) . ITs constructed with anti-CD30 moabs chemically linked to catalytically active toxins demonstrated specificity and potent antitumor activity against Hodgkin’s lymphoma cells in vitro and in mouse models (7 , 8) . A total of 12 patients with refractory relapsed Hodgkin’s lymphoma were treated with an IT constructed by conjugating the anti-CD30 moab BerH2 to Saporin-6 (Ber-H2-S6; Ref. 9 ). Rapid regression of tumor masses ranging from 50% to > 75% (lasting 2–4 months) were observed in ~50% of patients underlining the validity of CD30 as a target antigen in HD (10 , 11) . The major obstacles observed in this and other trials are unspecific toxicities, mainly related to the vascular leak syndrome, and the immunogenicity of the foreign proteins resulting in only a limited number of applications (12 , 13) .

By using phage display technology, we generated several Hodgkin’s lymphoma-specific scFvs, which were then genetically fused to ETA’ (14) . The rITs recovered from periplasmic space of E. coli grown under osmotic stress conditions in the presence of compatible solutes were highly functional in terms of specific in vitro binding and in vivo cytotoxic activity.

Humanized or human antibody fragments have been used to reduce the immune response against xenogeneic proteins and indeed attenuated immunogenicity in patients (15) . We reported recently on a recombinant fusion protein consisting of recombinant human CD30 ligand fused to ETA’ and showed the CD30-specific cytotoxicity of this monomeric protein (16) . Because human RNases are present in extracellular fluids, human plasma, and tissues, they might be less immunogenic when used as toxic component of ITs. Human Ang, a human plasma protein with 65% homology to RNase A (17) , was documented as a potent inhibitor of protein synthesis in cell-free extracts and when injected into Xenopus oocytes (18) . The Mr 14,000 single chain polypeptide is not cytotoxic toward a wide range of cultured cells but exhibits specific cytotoxic activity when fused to a ligand, which is internalized on binding to the target cell (19 , 20) .

In this paper, we report our results with the human CD30L coding region genetically fused to the human Ang gene. We demonstrate that this new and completely human rIT Ang-CD30L exhibits specific and effective destruction of CD30+ lymphoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Oligonucleotides, and Plasmids.
E. coli XL1-blue {supE44 hsdR17 recA1 endA1 gyr A46 thi relA1 lacF’[pro AB+ lacIq lacZ{Delta}M15 Tn10(tetr)]} was used for propagation of plasmids and E. coli BL21[DE3; F- ompT hsSB(rB-MB-) gal dcm DE3] as host for synthesis of rITs. Synthetic oligonucleotides were synthesized by Eurogentec (Seraing, Belgium). Plasmids were prepared by the alkaline lysis method and purified using plasmid kits from Qiagen (Hilden, Germany). Restriction fragments or PCR products were separated by horizontal agarose gel electrophoresis and extracted with Qiaex II (Qiagen). Cloning into plasmid vectors was performed by standard methods.

Plasmid Construction.
CD30L cDNA gene was released from plasmid pDC202 (21) by PCR using the oligonucleotides CD30LBack (CAC-TTG-GAT-CAG-TCA-ATT-TTC-CGT-CGT-CCG-gaa-ttc-CAG-AGG-ACG-GAC-TCC-ATT-CCC-AAC-TCA-CCT; underlined: EcoRI consensus; double underlined: 3'-Ang region) and CD30LFor (cgg-cgg-ggt-acc-TTA-GTC-TGA-ATT-ACT-GTA-TAA-GAA-GAT-GGA-CAA; underlined: KpnI consensus). The Ang coding region was similarly amplified using the oligonucleotides AngBack (tat-tat-aag-ctt-CAG-GAT-AAC-TCC-AGG-TAC-ACA-CAC-TTC-CTG; underlined: HindIII consensus) and AngFor (AGG-TGA-GTT-GGG-AAT-GGA-GTC-CGT-CCT-CTG-gaa-ttc-CGG-ACG-ACG-GAA-AAT-TGA-CTG-ATC-CAA-GTG; underlined: EcoRI consensus; double underlined: 5'-CD30L region). PCR was performed in 50-µl reaction mixtures containing 20 mM Tris-HCl (pH 8.3), 10 mM KCl, 2 mM MgCl2, 6 mM (NH4)2SO4, 0.1% Triton X-100, and 100 mM deoxynucleotide triphosphate, 50 nmol of each primer, and 1 unit plaque-forming unit DNA polymerase; 20–50 ng of template DNA was used. General cycling conditions: 94°C for 5 min, 60°C hold (hot start); 94°C for 1 min, 50°C for 3 min, and 72°C for 6 min (30x); and 72°C for 3 min in a Biometra Personal Cycler. Both fragments were joined together by SOE-PCR (22) , restricted with HindIII/KpnI, and finally inserted into a HindIII/KpnI-restricted expression vector pBR substituting for a modified ricin A-chain gene behind a enterokinase-cleavable His10-Tag (23) . The resulting pBMAng-CD30L plasmid was transformed into E. coli BL21(DE3).

Cell Culture.
All of the cell lines including the CD30+ Hodgkin-derived cell lines L540, L540cy (24) , HDLM-2 (25) , KM-H2 (26) , L1236 (27) , and CD30- -MyZ (28 ; kindly provided by B. Dörken, Berlin, Germany) were cultivated in complex medium (RPMI 1640) supplemented with 10% (v/v) heat-inactivated FCS, 50 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. All of the cells were cultured at 37°C in a 5% CO2 in air atmosphere, cultivated in complex medium (RPMI 1640), and supplemented with 10% (v/v) heat-inactivated FCS, 50 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. All of the cells were cultured at 37°C in a 5% CO2 atmosphere.

Periplasmic Expression and Purification of the Recombinant Ang-CD30L.
Recombinant ITs were expressed under the control of the IPTG-inducible T7lac promoter in E. coli BL21(DE3) as described recently (29) . Briefly, bacteria were grown overnight at 26°C in Terrific Broth (30) containing 50 µg/ml kanamycin and 0.5 mM ZnCl2. The culture was diluted 30-fold in 200 ml of the same medium. At an A600 of 2 it was supplemented with 0.5 M sorbitol, 4% NaCl, and 10 mM glycine betaine, and incubated at 26°C for and additional 30–60 min. Expression of rIT was induced by the addition of 2 mM IPTG at 26°C. Later (15 h), cells were harvested by centrifugation at 3,700 x g for 10 min at 4°C. For all of the next steps, tubes were kept on ice. The bacterial pellet was centrifuged and its wet weight determined. Cells were frozen at -196°C. After thawing, the cells were resuspended in 75 mM Tris-HCl (pH 8), 300 mM NaCl, 1 capsule of protease inhibitors/50 ml (Complet, Roche Diagnostics, Mannheim, Germany), 5 mM DTT, 10 mM EDTA, and 10% (v/v) glycerol, and sonicated six 6 times for 30 s at 200 W. The periplasmic fraction was recovered after centrifugation at 21,000 x g for 30 min at 4°C and transferred to 75 mM Tris-HCl (pH 8), 1 M NaCl, and 10% glycerol using Hitrap desalting columns (Pharmacia, Freiburg, Germany). rIT was partially purified by immobilized metal-ion affinity chromatography that contained nickel-nitriloacetic acid chelating Sepharose (Qiagen) on a BioLogic workstation (Bio-Rad, München, Germany). Bound protein was eluted with 250 mM imidazole in 75 mM Tris-HCl (pH 8), 1 M NaCl, and 10% glycerol. Fractions containing the Ang-CD30L were pooled, concentrated by ultrafiltration, and finally purified using SEC with Bio-Prep SE-100/17 columns (Bio-Rad) on the BioLogic workstation. Protein was eluted with PBS (pH 7.4) and 1 M NaCl, analyzed by SDS-PAGE, quantified by densitometry (GS-700 Imaging Densitometer; Bio-Rad) after Coomassie staining in comparison with BSA standards and verified by Bradford assays (Bio-Rad).

Enterokinase Cleavage.
To remove the NH2-terminal His10-Tag, highly specific cleavage of rIT (behind asp-asp-asp-asp-lys) by recombinant enterokinase (1 unit/50 µg rIT) was performed in duplicate using the enterokinase cleavage kit (Novagen, Abingdon, United Kingdom) as described by the manufacturer. Functional rIT was finally purified using SEC on the BioLogic workstation by separation in PBS (pH 7.4). Purified protein was analyzed by SDS-PAGE and quantified by densitometry (GS-700 Imaging Densitometer; Bio-Rad) after Coomassie staining in comparison with BSA standards and verified by Bradford assays (Bio-Rad).

SDS-PAGE and Western Blotting.
SDS-PAGE and Western blotting were performed as described (31) . Ang-CD30L was detected by anti-Ang-biotin moab (Sigma Chemical Co.). Bound antibody was stained with an alkaline-phosphatase-conjugated antimouse-IgG moab (Sigma Chemical Co., Deisenhofen, Germany) and a solution of Tris-HCl (pH 8.0) and 0.2 mg/ml naphtol-AS-Bi-phosphate (Sigma Chemical Co.) plus 1 mg/ml Fast-Red (Serva, Heidelberg, Germany).

Sandwich-ELISA.
The binding activity of Ang-CD30L was determined by CD30 receptor Sandwich-ELISA as documented previously (DAKO CD30 kit). The Microwell Strips was precoated with mouse moab to human CD30 receptor. After washing, 30 units/ml of CD30 (in Tris-buffered saline containing 0.5% BSA and 0.05% Tween 20) were incubated for 2 h at room temperature. After washing, samples were incubated for 2 h. Unbound IT was removed, wells were thoroughly washed and subsequently incubated with anti-Ang moab-biotin (R&D Systems) for 1 h. After washing, 100 µl of Streptavidin-POD (Roche Diagnostics, Mannheim, Germany; 1:5000 in Tris-buffered saline containing 0.5% BSA and 0.05% Tween 20) were added and samples incubated for 1 h at room temperature. Bound Ang-CD30L was detected by addition of 100 µl O-phenylenediamine-dihydrochloride (Sigma Chemical Co.) after incubation for 1 h. Absorbance at 405 nm was measured with an ELISA reader (MWG Biotech, Ebersberg, Germany). Native moabs were used as controls.

Flow Cytometry Analyses.
Cell binding activity of Ang-CD30L expressed in E. coli BL21(DE3) was evaluated by flow cytometry as described (31) . Briefly, cell suspensions containing 5 x 106 L540cy cells/ml were washed in PBS containing 0.2% w/v BSA and 0.05% w/v sodium azide (PBS/BSA/N3-) and incubated for 30 min in blocking solution (10% BSA in PBS/N3-). After one wash in PBS/BSA/N3-, the cells were incubated in 100 µl of the Ang-CD30L protein extract for 2 h at 4°C. The cells were washed again three times and treated with anti-Ang moab (R&D Systems) for 1 h. After three more washes, the cells were incubated with FITC-labeled goat-antimouse immunoglobulin for 1 h at 4°C. The cells were subsequently washed three times and analyzed on a FACScan (Becton Dickinson, Heidelberg, Germany). For competition, 10 µg/ml CD30L-CD8-Biotin (Ancell, Bayport, MN) or moab Ki-4 were used; Ang-CD30L-his bound to L540 cells was detected by moab anti-penta-His (Qiagen) and FITC-labeled antimouse IgG.

RNase Assay.
To measure the ribonucleolytic activity of human Ang, a tRNA assay was performed as described (32) . In this study, conditions were optimized as followed. Yeast-tRNA (0.6 µg; Sigma Chemical Co.) were incubated with enterokinase-cleaved and molecular size-purified Ang-CD30L in 30 mM Tris-HCl (pH 7.5) containing 30 mM NaCl in a total volume of 40 µl. After 20 h at 4°C, the reaction was terminated by addition of sample buffer consisting of 48% formaldehyde, 48% glycerol supplemented with 0.25% bromphenol blue and 2% 1 M sodium phosphate (pH 7.5). The samples were loaded onto a 1% agarose gel containing 20 mM MOPS, 5 mM NaOAc, 1 mM EDTA, 65 mM formaldehyde (pH 7; Qiagen) and electrophoresed in gel running buffer (20 mM MOPS,), 5 mM NaOAc, 1 mM EDTA, 65 mM formaldehyde, and 0.1% EtBr (pH 7).

Colorimetric Cell Proliferation Assay.
The cytotoxic effect of Ang-CD30L of the target cells was determined by measurement of metabolization of XTT to a water soluble orange formazan dye as published previously (29) . Various dilutions of the recombinant toxin were distributed in 100-µl aliquots in 96-well plates. Target cells (2–4 x 104) in 100-µl aliquots of complete medium were added, and the plates were incubated for 48 h at 37°C. Subsequently, the cell cultures were pulsed with 100-µl fresh culture medium supplemented with XTT/phenazine methosulfate (final concentrations of 0.3 mg and 0.383 ng, respectively) for 4 h. The spectrophotometrical absorbances of the samples were measured at 450 and 650 nm (reference wavelength) with an ELISA reader (MWG Biotech). The concentration required to achieve an IC50 of protein synthesis relative to untreated control cultures was calculated by nonlinear regression using SPSS. All of the measurements were done in triplicate. Competition experiments were performed on L540Cy cells in the presence or absence of 10 µg/ml purified Ki-4 moab as described (14) or 10 µg/ml CD30L-CD8 chimeric protein (Ancell) as competitors for a dilution series of Ang-CD30L.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction and Expression of Ang-CD30L.
PCR-amplified and assembled Ang-CD30L cDNA was inserted into the expression vector pBR, thus replacing the ricin A-chain gene (Fig. 1, a–c)Citation . The pBR expression vector is a derivative of the pET27b plasmid and contains an IPTG-inducible lac operator, a pelB signal peptide followed by an enterokinase-cleavable His10-Tag (23) . Successful cloning was verified by sequence analyses.



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  		Fig. 1. Cloning and expression of Ang-CD30L. a, schematic structure of the Ang-CD30L insert in the E. coli expression plasmid. The expression module is composed of the signal peptide of the pectate lyase of Erwinia carotovora (pelB), the IPTG inducible lac operator, a synthetic and enterokinase-cleavable His10 cluster (His-Tag), the human Ang gene, and the CD30L coding region. b, plasmid pBRAng-CD30L for the expression of the rIT in E. coli. The plasmid contains the expression module, a kanamycin resistance gene (kanr), an E. coli origin of replication (pBR322 origin), an M13 origin of replication (f1 origin), and the lactose repressor gene (lacI). The toxic moiety (Ang) is genetically fused to the binding structure (CD30L). c, Ang-CD30L open reading frame after SOE-PCR and restriction with HindIII/KpnI (Lane 1, Ang-CD30L (~980 bp); M, 100 bp ladder. d, Ang-CD30L protein after enterokinase cleavage and SEC as documented after SDS-PAGE and Comassie staining; M, Prestained Marker (Bio-Rad); 1, Ang-CD30L (34 kDa).

 
After cultivation of transformed E. coli BL21(DE3) under osmotic stress conditions in the presence of compatible solutes, recombinant foreign protein was functionally directed into the periplasmic space. The Mr 34,000 Ang-CD30L was purified by combinations of immobilized metal-ion affinity chromatography followed by enterokinase cleavage and SEC (Fig. 1d)Citation to a final minimum concentration of about 1 mg/4 g cell paste from 1 liter of bacterial shaking cultures (Fig. 2)Citation .



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  		Fig. 2. Functional activity of purified rITs after molecular size chromatography. a, elution profile monitored at 280 nm () combined with binding activity of the eluted fractions documented by Sandwich-ELISA (····). Captured recombinant human CD30 receptor was incubated with various concentrations of Ang-CD30L. Specifically bound IT was detected after incubation with polyclonal goat anti-Ang-biotin followed by Streptavidin-POD. Converted phosphatase substrate (O-phenylenediamine-dihydrochloride) was measured as absorbance at 405 nm. b, cell-binding activity of Ang-CD30L evaluated by flow cytometry analysis. L540cy cells were incubated with PBS (gray filling), His-tagged Ang-CD30L (black filling), enterokinase-cleaved Ang-CD30L (black line), or moab Ki-4 (doted line) for 60 min at 4°C. Cells were stained with mouse-anti-Ang moab and with goat-antimouse FITC-conjugated antibody. Immunofluorescence (FL1 channel) was measured by flow cytometry using a FACScan (Becton Dickinson).

 
Binding Properties of Ang-CD30L.
Binding properties of the enterokinase-cleaved Ang-CD30L against CD30+ target cell lines was measured by Sandwich-ELISA using captured soluble human CD30 receptor (Fig. 2a)Citation and flow cytometry. Binding properties of the recombinant protein against the native target antigen was documented by flow cytometry using an anti-Ang moab. Ang-CD30L bound to the CD30+ Hodgkin-derived cell line L540Cy but not to the CD30- HD-MyZ (Fig. 2b)Citation . CD30-specificity was documented by competitive flow cytometric experiments. Binding of Ang-CD30L against CD30+ L540 cells was inhibited by 10 µg/ml chimeric CD30L-CD8-Biotin (Fig. 3d)Citation or 10 µg/ml moab Ki-4 (Fig. 3e)Citation confirming this moab as a competitor for CD30L as described recently (33) .



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  		Fig. 3. CD30-specific binding activity of Ang-CD30L documented by competitive flow cytometric analyses. L540 cells were incubated with: a, Ang-CD30L; b, CD30L-CD8-biotin; c, moab Ki-4; d, Ang-CD30L competed with CD30L-CD8-biotin; and e, CD30L-CD8-biotin competed with moab Ki-4. For competition with 10 µg/ml CD30L-CD8-biotin (Ancell) or moab Ki-4 were used, Ang-CD30L-his bound to L540 cells was detected by moab anti-penta-His (Qiagen, Düsseldorf, Germany) and FITC-labeled antimouse IgG. Cells were stained with PBS as negative controls (gray curves and solid line in e).

 
RNase Assay.
Human Ang and RNase A (Sigma Chemical Co.) were used as positive controls to establish the RNase assay. The ribonucleolytic activity of enterokinase-cleaved Ang-CD30L is shown in Fig. 4Citation . The major product generated by endonucleolytic cleavage of tRNA was visualized after 1% denaturing agarose gel electrophoresis and EtBr staining. After 20 h of incubation at 4°C, enterokinase-cleaved Ang-CD30L showed ribonucleolytic activity against yeast tRNA (0.6 µg) at an absolute concentration of 150 ng.



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  		Fig. 4. Ribonucleolytic activity of Ang. Yeast-tRNA (0.6 µg; Sigma Chemical Co.) were incubated with enterokinase-cleaved and molecular size-purified Ang-CD30L in 30 mM Tris-HCl (pH 7.5), containing 30 mM NaCl in a total volume of 40 µl. After 20 h at 4°C, the reaction was terminated by addition of sample buffer consisting of 48% formaldehyde and 48% glycerol supplemented with 0.25% bromphenol blue and 2% 1 M sodium phosphate (pH 7.5). The samples were loaded onto a 1% agarose gel containing 20 mM MOPS, 5 mM NaOAc, 1 mM EDTA, and 65 mM formaldehyde (pH 7; Qiagen), and electrophoresed in gel running buffer (20 mM MOPS), 5 mM NaOAc, 1 mM EDTA, 65 mM formaldehyde, and 0.1% EtBr (pH 7). Lanes A–C, enterokinase-cleaved and molecular size-purified Ang-CD30L (150ng, 75 ng, and 37 ng); Lanes D, E, I, and J, negative controls; Lanes K–M, human Ang (75 ng, 35 ng, and 18 ng); Lanes F–H, RNase A (75 ng, 35 ng, and 18 ng).

 
Cytotoxic Activity in Vitro.
To characterize the cytotoxic activity of the rIT in vitro, we measured the proliferation of different target cells on incubation with different amounts or rIT. As shown in Fig. 5Citation , Ang-CD30L released by enterokinase-cleavage was cytotoxic to CD30+ L540 with a calculated median IC50 of 8 ng/ml. The fusion toxin also showed effective killing of all CD30-positive cell lines tested in parallel, including HDLM-2, KM-H2, L1236, L540, and L540Cy (Table 1)Citation . The CD30- Hodgkin-derived cell line HD-MyZ was not affected at recombinant protein concentrations of up to 10 µg/ml. His-tagged Ang-CD30L also did not exhibit any cytotoxic effect against L540 cells. In addition, the cytotoxicity against CD30+ cells was specifically inhibited by an excess of purified moab Ki-4 or CD30L-CD8 chimeric protein.



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  		Fig. 5. Growth inhibition of Hodgkin-derived cell lines after incubation with Ang-CD30L as documented by cell viability assays. a, L540 (CD30+) or HD-MyZ (CD30-) were treated with enterokinase-cleaved rIT, and their ability to metabolize the XTT to a water-soluble formazan salt (formed by mitochondrial dehydrogenase activity) was measured as absorbance at 450 and 650 nm (reference wavelength) in a cell viability assay (43) . Measurements were performed in triplicate. Results are presented as percentage of untreated control cells. b, cytotoxic activity of Ang-CD30L (····) on L540 and its competition with 10 µg/ml CD30L-CD8 ( ·· ) or moab Ki-4 () HD-MyZ (----) cells were used as negative controls. Measurements were again performed in triplicate.

 

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  		Table 1 Growth inhibition of Hodgkin-derived cell lines after incubation with Ang-CD30L as documented by cell viability assays

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we report the construction of a novel, completely human recombinant fusion toxin targeting CD30 receptor. For this purpose we fused the CD30L coding region to a modified human Ang. The major findings to emerge from our study are: (a) functional Ang-CD30L was directly isolated from the periplasmic space of E. coli cultured under osmotic stress conditions in the presence of compatible solutes by a combination of immobilized metal affinity and molecular size chromatography; (b) a free NH2-terminal region of the fusion protein was generated after enterokinase cleavage of the His10-Tag; (c) Ang-CD30L bound to CD30+ cells as documented by flow cytometry and CD30 receptor ELISA and binding was competed by the addition of moab Ki-4 or chimeric CD30L-CD8; and (d) the recombinant toxin exhibited specific cytotoxicity toward CD30 receptor-expressing Hodgkin-derived cell lines HDLM-2, KM-H2, L1236, L540, and L540Cy, but not against CD30-negative HD-MyZ cells. Different concentrations of CD30 receptor on the surface of the various Hodgkin-derived cell lines tested (34) , might be the reason for varying IC50s. CD30 specificity was again demonstrated by competition experiments.

One of the major problems identified in clinical trials with chemically linked ITs is the development of neutralizing antibodies against both the murine IgG and the toxic moiety resulting in a limited number of applications in about 40–60% of the patients (35 , 36) . This problem might, at least in part, be overcome by using recombinant DNA technology to construct smaller and less immunogenic ITs. Very recently, the first clinical trial with such a construct, anti-Tac(Fv)-PE38 (LMB-2), in patients with CD25+ hematological malignancies has been published suggesting a reduced antibody response (37 , 38) . To additionally diminish this immunogenicity, humanized antibody fragments or cytotoxic activities derived from human resources (39) might be used. Human CD30 ligand has been described recently as a type II membrane protein. Its COOH-terminal, extracellular subdomain shares sequence homologies with corresponding regions of tumor necrosis factor {alpha}, tumor necrosis factor ß, and CD40L (40) . Thus, we have used the putative human soluble CD30L portion as binding subdomain for the construction of a Pseudomonas exotoxin-based fusion protein (16) . Although ETA’ was genetically fused to the COOH-terminus of the CD30L, which normally remains free when displayed on the surface of eukaryotic cells, the protein showed CD30-specific binding and cytotoxic activity. Because the NH2 terminus is crucial to the cytotoxicity of RNases (39) , CD30L was in this study fused to the COOH-terminus of human Ang, thus inserting the extracellular spacer region of CD30L followed by the COOH-terminal receptor binding domain (41) . The periplasmically expressed, non-glycosylated recombinant fusion protein used in this experimental setup exhibited specific cytotoxicity after removal of the NH2-terminal His10-Tag. The calculated cytotoxic activity of Tag-free Ang-CD30L against L540 cells was three times higher (IC50 = 8 ng/ml) compared with CD30L-ETA’ (IC50 = 24 ng/ml) published recently (16) . Additionally, the concentration needed to achieve 50% inhibition of cell growth for Ang-CD30L is in a very similar range to the Pseudomonas exotoxin-based anti-CD30 IT Ki-4(scFv)-ETA’ (6 ng/ml; Refs. 29 , 42 ). Cross-linking of the CD30 target antigen does not seem to be necessary for the monomeric IT to be internalized. This phenomenon correlates with our data from recombinant CD30L-ETA’ and anti-CD30 scFvs fused to ETA’ as binding structures. Both binding activity and cytotoxic efficiency of the recombinant, monovalent fusion toxin might be additionally increased by creating polyvalent modifications thereby enhancing internalization efficacy by receptor mediated endocytosis.

In summary, we demonstrated the construction of a recombinant, tag-free fusion toxin, which specifically binds to the CD30 receptor. The use of modified recombinant human CD30L as a binding structure for recombinant immunotherapeutics against Hodgkin’s lymphoma and in combination with Ang will be additionally investigated for in vivo application in humans.


   ACKNOWLEDGMENTS
 
We thank Dr. Hans-Jörg Gruss for providing CD30L. We also thank Silke Drillich for cloning assistance and Gisela Schön for performing the toxicity assays.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Deutsche Forschungsgemeinschaft Grant SFB502 (to A. E. and S. B.). Back

2 To whom requests for reprints should be addressed, at Fraunhofer IME, Department of Pharmaceutical Product Development, Worringer Weg 1, 52074 Aachen, Germany. Phone: 49-241-80-28399; Fax: 49-241-871062; E-mail: barth{at}ime.fhg.de Back

3 The abbreviations used are: H-RS, Hodgkin/Reed-Sternberg; IT, immunotoxin; HD, Hodgkin’s Disease; ETA’, deletion mutant of Pseudomonas exotoxin; MOPS, 3-[N-morpholino]propanesulfonic acid; Ang, angiogenin; IPTG, isopropyl ß-D-thiogalactoside; moab, monoclonal antibody; EtBr, ethidium bromide; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; rIT, recombinant immunotoxin; scFv, single-chain variable fragment; SEC, size exclusion chromatography. Back

Received 10/30/00. Accepted 10/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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