Bioconjugate Cbem. 1995, 6, 666-672
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ARTICLES IL2-Ricin Fusion Toxin Is Selectively Cytotoxic in Vitro to IL2 Receptor-BearingTumor Cells Arthur Frankel,",' Edward Tagge,' J o h n Chandler,' Chris Burbage,+ Greg Hancock,' Joseph Vesely,$ a n d Mark Willinghams Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, South Carolina 29425. Received April 12, 1995@
Fusion toxins consist of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin toxin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure, and the years of experience with RTA chemical conjugates in clinical trials. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL2, another ADP tripeptide, and RTB. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL2-RTB protein was recovered a t high yields from day 5 insect cell supernatants, partially purified by affinity chromatography, and characterized. The recombinant product was soluble and immunoreactive with antibodies to RTB and IL2, bound asialofetuin and lactose, and reassociated with RTA. In the presence of lactose to block galactosebinding sites on RTB, the IL2-RTB-RTA heterodimer was selectively cytotoxic to IL2 receptor, bearing cells. Specific cytotoxicity could be blocked with IL2. Thus, we report a novel targeted plant toxin fusion protein with full biological activity.
INTRODUCTION
Ricin toxin, the 65 kDa heterodimeric glycoprotein from castor bean seeds, consists of a lectin B chain (RTBY disulfide linked to an enzymatic A chain (RTA)( I ) . Ricin intoxication of mammalian cells involves sequentially (a) RTB binding to P-galactosyl pyranoside groups on cell surface glycoproteins (21, (b) internalization by endocytosis (31, (c) transfer to the TR Golgi (41, (d) routing to a critical organelle, possibly the endoplasmic reticulum (51, (e) disulfide bond reduction with release of RTA (61,(0 translocation of RTA to the cytosol, and (g) catalytic inactivation of protein synthesis by hydrolysis and release of an adenine base from the elongation factor binding site of 26s rRNA in the 60s ribosomal subunit (7). A single molecule of ricin introduced into a cell can lead to cell death (8). Because of this extreme potency, a number of groups have attached RTA or modified ricin molecules to new ligands to achieve selective cell killing in vitro and in vivo and then used these immunotoxins systemically in patients with refractory neoplasms. The Fab' fragment of a murine monoclonal anti-CD22 antibody was coupled to chemically deglycosylated RTA and administered intravenously to patients with B-cell lymphomas (9). ~
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* Address correspondence to this author at the following address: Hollings Cancer Center Rm 306, MUSC, 171Ashley Ave., Charleston, SC 29425. Tel: 803-792-1450. Fax: 803-792-3200. Department of Medicine. * Department of Surgery. 5 Department of Pathology. Abstract published in Advance ACS Abstracts, September 15, 1995. Abbreviations: IL2, interleukin-2; RTB, ricin toxin B chain; RTA, ricin toxin A chain; IL2-RTB, fusion protein of interleukin-2 and ricin B chain. @
1043-1802/95/2906-0666$09.00/0
While 50% of patients with antigen on the tumor cell surfaces showed a partial response, significant doselimiting toxicity to vascular endothelium was observed. The small size (80 000 Mr) of the conjugate may have facilitated tumor penetration, but no studies of drug distribution were done. Mouse antibody to CD22 was coupled to chemically deglycosylated RTA and administered to lymphoma patients (10). Both partial and complete responses were observed, but again vascular leak syndrome was the dose-limiting toxicity. Higher peak concentration of immunotoxin in the serum, longer 2'112, and larger AUC correlated with vascular injury. Murine monoclonal antibody to CD5 ws thiolated and coupled to RTA and administered to patients with chronic lymphocytic leukemia (11). At doses up to 16 mg/mz, no immunotoxin could be demonstrated a t extravascular sites. Mouse antibody to a 55 kDa epithelial cell surface glycoprotein was derivatized and conjugated to recombinant RTA and infused into patients with metastatic breast carcinoma (12). Four out of five patients developed anti-mouse Ig and anti-RTA antibodies. Ricin toxin was chemically blocked with an affinity ligand and crosslinked to an anti-CD19 monoclonal antibody (13). After administration to patients with lymphoma, different preparations of immunotoxin produced different degrees of hepatocyte damage. Further analysis revealed product heterogeneity with the more toxic species having one to two affinity ligands per ricin and the less toxic species with three affinity ligands per ricin. Thus, pharmacologic properties of drug heterogeneity, poor drug penetration, normal tissue toxicity, and immunogenicity have reduced the therapeutic index (ratio of dose producing toxicity1 dose producing clinical efficacy)in clinical trials with RTA or blocked ricin immunotoxins. Methods have been 0 1995 American Chemical Society
112-Ricin Fusion Toxin sought to improve the pharmacologic properties of these protein therapeutics. One approach has been to genetically engineer the toxin and ligand into a single well-defined molecule. Normal cell binding portions of diphtheria toxin and Pseudomonas exotoxin have been deleted and replaced with growth factors and single chain Fv’s (14, 15). Clinical trials with TGFa-PE40 and DAB3891L2bacterial fusion toxins have shown excellent tolerance (minimal side effects or toxicities) and significant clinical activity (16, 17). A fusion protein consisting of basic fibroblast growth factor and the ribosome-inactivating protein saporin (FGF-SAP) has been expressed in E . coli (18). FGF-SAP demonstrated potent specific cell cytotoxicity M) and inhibited growth of B16 (ID50 = 5 x melanomas both in subcutaneous implants and lung metastases in mice. Surprisingly, few studies have been done directly comparing fusion toxins with chemical conjugates. Lappi reported no difference in vitro or in vivo between FGF-SAP linked by a cross-linker or by a n amide bond (18).Kreitman compared anti-Tac-PE with the single chain fusion toxin anti-Tac(Fv)-PE40 (19,20). In vitro and in vivo, the fusion toxin was approximately 1log more active. An additional advantage of the fusion toxin was the reagent homogeneity. Attempts have been undertaken to produce genetically engineered ricin or RTA fusion proteins, in part because of the extensive clinical experience with RTA and blocked ricin immunotoxins showing safety in patients (9-13) and, in part, due to the potential use of ricin fusion toxins in patients who have developed resistance to diphtheria toxin or Pseudomonas exotoxin fusion proteins. RTA inactivates protein synthesis by specifically depurinating a conserved adenosine in the 60s ribosomal subunit, while diphtheria toxin and Pseudomonas exotoxin act by ADP-ribosylating EF-2 ( 7 ) . Thus, malignant cells are unlikely to show cross-resistance to both plant and bacterial toxins. Further, the amino acid sequence and three-dimensional structure of ricin is distinct (21) from the diphtheria toxin and Pseudomonas exotoxin, and thus, antibodies to one toxin do not react with the other toxins (unpublished observations). An RTA-diphtheria toxin loop-Staphylococcal protein A fusion protein was expressed in E . coli, enzymatically cleaved with trypsin, mixed with antibody, and exposed to antigen positive cells (22). Selective cytotoxicity was demonstrated. However, the fusion toxin antibody conjugate had unfavorable properties. The disulfide loop was exposed on the surface of the conjugate and readily reduced. The RTAdiphtheria toxin-protein A-immunoglobulin conjugate was very large (’200 000 Da). The conjugate was heterogeneous due to varying sites of protein A-immunoglobulin binding. Subsequently, tripartate fusion proteins were produced in E . coli with IL2-diphtheria toxin loop-RTA or IL2-factor Xa recognition sequence-RTA (23). Proteolytic cleavage with trypsin or factor Xa released the IL2 ligand and toxin without recovery of disulfide-linked product. Uncleaved chimeras showed no cytotoxicity to IL2 receptor-bearing cells. Finally, a factor Xa-specific site was introduced into the linker sequence of proricin and the modified proricin expressed in Xenopus oocytes (24). Although recombinant mutant proricin was produced, yields were in the nanogram range and protease sensitivity was low. Further, no fusion proteins with IL2 binding specificities were made. We chose an alternative approach for genetic engineering of ricin. Previous studies document the need for an accessible disulfide bridge between ligand and RTA (6, 22). Consequently, we chose to produce recombinant RTB fusion proteins with novel ligands and reassociate
Bioconjugafe Chem., Vol. 6,No. 6, 1995 667
the molecule with RTA to recreate the natural disulfide bridge between RTA and RTB. The ligand was attached to the N-terminus of RTB based on our previous experience with a n oligohistidine tag and the X-ray crystallographic structure of ricin (21,251. In the present study, we synthesized DNA encoding the GP67A leader peptide, IL2, and RTB. Recombinant protein was expressed and secreted from insect cells. The fusion molecule was purified, reassociated with plant RTA, and tested for selective cytotoxicity to IL2 receptor positive cells in the presence of lactose. EXPERIMENTAL PROCEDURES
Materials. Restriction endonucleases and T4 ligase were obtained from Promega (Madison, WI). [32PldCTP, [35SldATP,L3H1leucine were obtained from Amersham (Arlington Heights, IL). Rabbit antiricin antibody, alkaline phosphatase conjugated goat anti-(rabbit IgG), alkaline phosphatase conjugated goat anti4mouse IgG), asialofetuin, a-lactose, and other chemicals were from Sigma (St. Louis, MO). EX-CELL400 medium was obtained from JRH Scientific (Lexena, KS). Sf9 insect cells, TMNFH medium, BaculoGold DNA, and pAcGP67A transfer fector were from PharMingen (San Diego, CAI. Prep-A-Gene DNA and plasmid purification matrices, low molecular weight prestained protein standardards, nitrocellulose paper, and other reagents for protein analysis were obtained from BioRad (Hercules, CAI. The Sequenase kit for dideoxy sequencing was obtained from USB (Cleveland, OH). The Random Primer labeling kit was obtained from Stratagene (La Jolla, CA). Purified P2, P8, and P10 murine monoclonal antibodies to RTB and purified aBR12 murine monoclonal antibody to RTA were gifts of Dr. Walter Blattler, ImmunoGen (Cambridge, MA). RPMI1640 media, leucine-free RPMI1640, penicillin, streptomycin, Dulbecco’s PBS, fetal bovine serum, and dialyzed fetal bovine serum were obtained from GIBCO BRL (Grand Island, NY). 3M Emphaze Biosupport medium AI31 azlactone functionality bis-acrylamide and lactosyl acrylamide were obtained from Pierce (Rockford, IL). The alkaline phosphatase Vectastain kit for Western blots was obtained from Vector Laboratories (Burlingame, CA). EL4 plates and round-bottomed and flat-bottomed 96-well plates were from Costar (Cambridge, MA). Plant RTB, ricin, and RTA were obtained from Inland Laboratories (Austin, TX). Constructionof Plasmid. pDW27 plasmid containing DAB3dL2 DNA was a gift of Dr. John Murphy (Boston University) (27). PCR was performed with pDW27 plasmid encoding DAB3s91L2 and the 5’ oligonucleotide 5’-GCAGCATCAGGATCCCGCACCTACTTCTA-GCTCT-3’, which introduces a BamHI site followed by a CCC proline codon followed by DNA encoding the first six codons of IL2. The 3‘ oligonucleotide was
fj’-AGCTGCAGATGGAT-CGCGGTCAGGGTAGAGATGAT-3’, which contains the last six codons of IL2 followed by a GC and a BamHI site. The PCR product, which provided a n IL2 DNA BamHI cassette maintaining the proper reading frame a t both ends with the GP67A leader and RTB, was purified on a silica matrix (Prep-A-Gene, BioRad), digested with BamHI, and subcloned in BamHI restricted pAcGP67A-RTB plasmid (24). The final vector was double-stranded dideoxy sequenced by the Sanger method with Sequenase reagents (USB). One liter cultures of transformed E. coli were subjected to alkaline lysis, and the plasmid was purified by cesium chloride density gradient centrifugation. Expression of Fusion Toxin. A 2 x lo6 Sf9 sample of S . frugiperda ovarian cells maintained in TMNFH medium supplemented with 10% fetal calf serum was
668 Bioconjugate Chem., Vol. 6,No. 6,1995
cotransfected with pAcGP67A-IL2-RTB DNA (4 pg) and 0.5 pg of BaculoGold AcNPV DNA following the recommendations of the supplier (PharMingen). At 5 days post-transfection, medium was centrifuged and the supernatant tested in a limiting dilution assay with Sf9 cells and dot blots with random primer 32P-dCTPlabeled RTB DNA (Stratagene Prime-It kit) as previously described (28). Positive wells were identified and supernatants reassayed by limiting dilution until all wells up to dilution were positive. Two rounds of selection were required. Recombinant virus in the supernatant was then amplified by infecting Sf9 cells at a multiplicity of infection (moi) of 0.1, followed by collection of day 7 supernatants. Recombinant baculovirus was then used to infect 2 x lo8 Sf9 cells at a n moi of 5 in 150 mL of EX-CELL400 medium (JRH Scientific) with 50 mM a-lactose in spinner flasks. Media supernatants containing IL2-RTB were collected day 6 postinfection. Mfication of IL2-RTB. Media supernatants were adjusted to 0.01% sodium azide and maintained through all purification steps at 4 "C. The supernatants were concentrated 15-fold by vacuum dialysis, centrifuged at 3000g for 10 min to remove precipitate, dialyzed against 50 mM NaC1, 25 mM Tris pH 8, 1 mM EDTA, 0.01% sodium azide, and 25 mM a-lactose (NTEAL), ultracentrifuged at lOOOOOg for 1h, and bound and eluted from a P2 monoclonal antibody-acrylamide matrix as previously described (28). P2 is a n anti-RTB monoclonal antibody. The affinity matrix was prepared using Ultralink azalactone functionality bis-acrylamide following the recommendations of the manufacturer. Recombinant protein was adsorbed to the column in NTEAL, washed with 500 mM NaC1,25 mM Tris pH 8 , l mM EDTA, 0.1% Tween 20,0.01% sodium azide, and 25 mM a-lactose, and eluted with 0.1 M triethylamine hydrochloride pH 11. The eluant was neutralized with 1/10 volume 1M sodium phosphate pH 4.25 and stored at -20 "C until assayed. Four preparations were made. Molecular Weight Determination. Aliquots of IL2RTB, recombinant RTB, plant RTB (Inland Laboratories), and prestained low molecular weight standards (BioRad) were run on a reducing 15% SDS/PAGE, stained with Coomassie Blue R-250, dried between cellophane sheets, and scanned on a IBAS 2000 automatic image analysis system (Kontron, Germany). Immunological Characterization. Aliquots of IL2RTB, bacterial IL2 (Chiron), wild-type recombinant RTB, plant RTB, and prestained low molecular weight protein standards were run on a reducing 15% SDS-PAGE, transferred to nitrocellulose, blocked with 10% Carnations nonfat dry milWO.l% BSNO.l% Tween 20, washed with PBS plus 0.05%Tween 20, reacted with either 1:400 rabbit antibody to ricin (Sigma) or 1:lOO mouse monoclonal antibody to IL2 (Genzyme), rewashed, incubated with alkaline phosphatase conjugated goat anti-(rabbit IgG) or anti-(mouse IgG) (Sigma), washed again, and developed with the Vectastain alkaline phosphatase kit (Vector Laboratories). Costar EIA microtiter wells were coated with 100 pL of 5 pg/mL of monoclonal antibody P2, P8, or P10 reactive with RTB (gifts of Dr. Walter Blattler, ImmunoGen) or monoclonal antibody to IL2 (Genzyme),washed with PBS plus 0.1% Tween 20, blocked with 3% BSA, rewashed, and incubated with samples of IL-2-RTB or plant RTB, rewashed, reacted with 1:400 rabbit antibody to ricin, washed again, incubated with 15000 alkaline phosphatase conjugated goat anti-(rabbit IgG), rewashed, developed with 1 mg/mL of p-nitrophenyl phosphate in diethanolamine buffer pH 9.6, and read on a BioRad 450 Microplate reader at 405 nm.
Frankel et al. A
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Figure 1. Fifteen percent reducing SDS-PAGE of IL2-RTB.
(A)Coomassie stained. Lane 1: low molecular weight prestained BioRad protein standards as marked by arrows and are 20 000, 28 500, 34 400, 52,000, 86 000, and 112 000 daltons; lane 2: IL2-RTB. (B) Immunoblot using rabbit anti-ricin antibody. Lane 1: low molecular weight protein standards marked by arrows as above. Lane 2: IL2-RTB. (C)Immunoblot using mouse anti-IL2 antibody. Lane 1: low molecular weight standards. Lane 2: IL2-RTB. Measurement of Lectin Activity. Asialofetuin (1 pg/mL) was bound to Costar EIA plate wells, and a n ELISA was performed as previously detailed with samples of IL2-RTB and plant RTB (28). Samples of freshly reduced IL2-RTB were diluted in 50 mM NaCl/25 mM Tris pH 8/1 mM EDTA/O.Ol% sodium azide (NTEA), loaded on a lactosyl acrylamide matrix (Pierce), and washed with NTEA, and the lactose binding protein was eluted with NTEA plus 50 mM lactose. Fractions were assayed for RTB immunoreactive material using the P2 antibody ELISA described above. Formation of Recombinant Heterodimer. Thirty pg of IL2-RTB was mixed with 90 pg of plant RTA in a total volume of 1mL of PBS and then shaken overnight at room temperature. The reaction mixture was then analyzed by a ricin ELISA previously described (25). Reassociated mixtures were also analyzed by nonreducing SDS-PAGE followed by immunoblots with P10 antiRTB monoclonal antibody and monoclonal antibody to IL2. Densitometric scanning with the automatic image analysis system was done to quantify the shift of immunoreactive material from 50 to 80 kDa. Cytotoxicity to Mammalian Cells. Measurement of protein synthesis inhibition by ricin, IL2-RTB-RTA, and DAB3891L2(14) in cultured cells was done as previously described using HUT102 human T leukemia cells bearing the high affinity IL2 receptor, CEM human T leukemia cells bearing the intermediate affinity IL2 receptor, and OVCAR3 human ovarian carcinoma cells lacking the IL2 receptor. All three cell lines were obtained from the American Type Culture Collection (Rockville, MD). HUT102 cells intermittently release HTLV-1 and should be handled with care. All assays were performed in triplicate. In some experiments duplicate samples were incubated in the presence of 20 ,ug/mL of IL2 (Chiron). The ID50 was the concentration of protein which inhibited protein synthesis by 50% compared with control. A total of 1.5 x lo4 HUT102 cells were placed in sterile Eppendorf tubes at 4 "C in 100 pL of leucine-poor RPMI1640 10% dialyzed fetal calf serum 60 mM a-lactose with or without 20 pg/mL of IL2. Dilutions of IL2-ricin and ricin at varying concentrations were added in identical medium with or without IL2 and incubated at 4 "C for 30 min. Cells were pelleted at 2000g for 5 min, washed once with leucine-poor RPMI1640 10% dialyzed fetal calf serum 60 mM a-lactose, resuspended in 150 pL of the same medium, and incubated at 37 "C in 5%COz for 24 h. [3H]leucinewas added as above, and 4 h later cells were harvested with the PhD cell harvester
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Bioconjugate Chem., Vol. 6,No. 6, 1995 669
112-Ricin Fusion Toxin
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2 3 4 5 Figure 2. Fifteen percent nonreducing SDS-PAGE of IL2-RTB-RTA. (A) Immunoblot using mouse monoclonal antibody P10 to RTB. Lane 1: low molecular weight standards. Lane 2: IL2-RTB. Lane 3: IL2-RTB-RTA. Lane 4: RTA-plant RTB. Lane 5: RTA-recombinant RTB. (B) Immunoblot using mouse monoclonal antibody to IL2. Lane 1: low molecular weight standards. Lane 2: IL2-RTB. Lane 3: IL2-RTB-RTA. Lane 4: RTA-plant RTB. Lane 5: RTA-recombinant RTB. Densitometry of gel B confirmed 85% of the RTA immunoreactive material migrated at 80 kDa afker reassociation.
and incorporated [3H]leucinemeasured in a liquid scintillation counter. Blocking of selective cytotoxicity was estimated by comparing the ID50of toxins in the presence or absence of IL2 lactose. There was no purification step after heterodimer reassociation. The free RTA concentration at the highest concentration of heterodimer in the assay (5 x M) was M. On all three cell lines, the ID50for free RTA was (2-3 x M) (unpublished results). Thus, in the range of heterodimer IDSO'S (10-10-10-12 M), the free RTA concentration (2 x M) should not produce cytotoxicity. M to 2 x
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Yield and purity of IL2-RTB. Material post-affinity chromatography was subjected to SDS-PAGE and Coomassie staining (Figure 1A). Over 80%of the protein migrated as a single band at 50 kDa. On the basis of absorbance at 280 nm (1mg/mL produced a n OD280 = 1.4) and densitometry, 1 mg/mL fusion protein was secreted per liter of insect cell culture. The major contaminants at 55, 60, and 90 kDa were not reactive with murine antibodies against RTA or IL2 and appeared in media of cells after lysis from wild-type AcNPV virus. They presumably represent insect cell derived proteins. Individual batches of IL2-RTB of about 150 ,ug were recovered from 150 mL of infected insect cell supernatants and were each adequate for all the studies undertaken. Immunologic Cross-Reactivity. Immunoblots demonstrated reactivity with the same 52 kDa band using anti-RTB or anti-IL2 antibodies (Figure lB,C). Interestingly, weaker bands of approximately 10- 15% intensity relative to the 52 kDa band were observed at 35 kDa reactive with anti-RTB and 20 kDa reactive with antiIL2 suggesting partial proteolysis occurred either intracellularly or in the medium. The site(s) of cleavage are unknown, but the size of the fragments and their specific reactivities with antibodies suggest the predominant site of cleavage is between the RTB and IL2 domain. This is not unusual for chimeras, as the secondary structure is likely to be least between domains. Antibody ELISA's demonstrated similar reactivity of IL2-RTB and plant RTB with anti-RTB monoclonal antibodies. Relative to plant RTB, the monoclonal antibody P2 reacted 66% as well with IL2-RTB. P8 and P10 monoclonal antibodies reacted 100% as well with IL2-RTB molecules as with plant RTB. Anti-IL2 antibody reacted with at least 70%of the RTB immunocrossreactive molecules on a molar basis. Lectin Activity. a-Lactose was coupled through the 6-hydroxyl groups of the glucosyl moiety to acrylamide, and binding of lectins was assessed. Plant RTB bound 100% to immobilized lactose, while 80% of recombinant wild-type RTB could be bound and eluted from lactosyl
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Table 1. Cell Clvtotoxiciw of Toxins to Various Cell Lines Drotein ricin IL2-RTB-RTA DAB3891L2
HUT102
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(5 f 2) x 10-10 (2 f 1)x 10-10 (5 f 3) x 10-10 (2 f 1)x 10-l' (6 f 2) x 10-l' (4 f 1)x (2 f 1)X (5 f 3) X lo-'
a 2 x lo4 cells in 150 pL of leucine-free RPMI1640 plus 60 mM a-lactose were combined with dilutions of toxins for 24 h a t 37 "C/5% CO2. 1pCi/well 3H leucine in 50 pL of the same media was added for 4 h a t 37 "C/5% C02. Cells were harvested with a PhD cell harvestor on glass fiber mats, dried, and counted in Econofluor in a LKB liquid scintillation counter. ID50 was the concentration of toxin reducing protein synthesis by 50%. Each assay perfomed three times. Standard deviations shown with mean.
acrylamide. Thirty percent of IL2-RTB attached to the matrix and eluted with 50 mM a-lactose. Asialofetuin consists of the bovine serum protein fetuin from which the terminal sialic acids have been chemically removed, exposing galactosyl residues. Binding of IL2RTB to asialofetuin adsorbed on microtiter wells provides a n independent assay of galactose binding affinity. The asialofetuin ELISA demonstrated that IL2-RTB bound immobilized asialofetuin 59% as well as plant RTB and wild-type recombinant RTB. Both the lactose binding and asialofetuin binding assays suggest the fusion molecule retains much of the lectin activity of wild-type recombinant RTB. The slight reduction in binding affinity may be secondary to steric hindrance by IL2 or misfolding affecting one or more sugar-combining subdomains. This residual lectin property is not desirable for a n in vivo therapeutic molecule. Reassociation with RTA. IL2-RTB provides a ligand function and coupling function for the fusion toxin, but the polypeptide must be linked to RTA for cytotoxicity. RTB has numerous amino acid residues which interact with RTA and promote both stabilization of the toxic heterodimer and protection of the intersubunit disulfide bridge (21). Provided the IL2 amino acid residues do not lead to misfolding or steric hindrance of the interface sidechains, reassociation of IL2-RTB with RTA should occur spontaneously at concentrations of M or higher of each component (26). Under the reaction M, PBS, room temperature, room air), conditions ( 50% reassociation was achieved with plant RTA and either plant or recombinant RTB. Under identical conditions, 60% reassociation of IL2-RTB occurred based on ricin ELISA and immunoblots with anti-RTB antibody (Figure 2). Cell cytotoxicity. Cytotoxicities of recombinant proteins and plant ricin for different cell lines are shown in Table 1. IL2-RTB alone was nontoxic (ID50> M) for all the cell lines tested. Ricin and IL2-ricin showed some toxicity (ID50= 1-6 x 10-lo M) to IL2 receptor
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negative cell lines in the presence of 60 mM a-lactose (Figure 3B,C). This residual toxicity is due to the presence of lectin binding sites on ricin and IL2-ricin that are incompletely blocked by 60 mM a-lactose. In contrast, IL2-ricin but not ricin was much more toxic to the IL2 receptor-positive HUT102 cells (ID50 = 4 x M) in the presence of lactose (Figure 3A). The in vitro therapeutic window with lactose in the media was 125fold. The control fusion toxin, DAB389IL2, also showed selective toxicity to IL2 receptor-bearing cells. Since DAB38gIL2lacks residual normal cell binding domains, the toxicity to nonreceptor bearing cells was considerably less (ID5o's = (5-15) x M). If the increased sensitivity of HUT102 cells to IL2ricin in the presence of lactose is due to receptor-specific binding, we should be able to block binding in the presence of excess IL2. HUT102 cells were exposed to IL2 (20 pglmL) and 60 mM lactose or lactose alone with varying concentrations of IL2-ricin or ricin for 30 min a t 4 "C. After cells were washed and then incubated overnight a t 37 "C/5%C02, cellular protein synthesis was measured by incorporation of [3Hlleucine. Each assay was repeated three times. The ID50of ricin with standard M in the presence of IL2 deviation was (3 f 2) x M in the absence of IL2. In contrast, and (6 f 3) x the ID50 of IL2-ricin with standard deveiations was (5 f 4)x M without IL2 and (3 f 2) x 10-lo M in the presence of excess IL2. The slightly lower specific toxicity of IL2-ricin under these conditions was due to the shortened exposure time. DISCUSSION
We report the construction and expression of a novel ricin fusion protein in significant yields. The baculovirus expression system has been previously used to independently express human IL2 and ricin toxin B chain (28, 29). We now describe the expression of the hybrid protein, IL2-RTB. Other chimeric eukaryotic proteins have been successfully expressed in insect cells using recombinant baculovirus including a fusion of the F and HN glycoproteins of human parainfluenza virus type 3 and a single chain monoclonal antibody composed of the variable domain of the light chain connected through AGQGSSV to the variable domain of the heavy chain (30, 31). In each case, the fusion protein needed a signal sequence for segregation into the endoplasmic reticulum with subsequent proper folding, disulfide bond formation, and glycosylation. The folding of each portion of IL2RTB was facilitated by attachment of IL2 to the Nterminus of RTB. The x-ray crystallographic threedimensional structure of ricin shows the N-terminus of RTB participates to a small degree in folding of RTB and interaction with RTA (21).Our results extend our earlier observation that a n oligohistidine tag attached to the N-terminus of RTB preserved folding for both the oligohistidine peptide and RTB (25). These observations provide the foundation for a general strategy of producing ricin fusion proteins by attachment of novel ligands to the RTB N-terminus. Evidence in addition to secretion, solubility and yield that IL2-RTB had proper folding of both the IL2 and RTB domains was the similar immunological reactivity of soluble IL2-RTB, RTB, and IL2 with antibodies to IL2 and RTB. Anti-IL2 antibody bound IL2 and IL2-RTB comparably, and anti-RTB antibodies bound RTB and IL2-RTB approximately equally (see Results). TheELISA assay format permitted quantitative measurements of soluble proteins. IL2-RTB retained most of the galactose-binding activity of plant and wild-type recombinant RTB. The X-ray
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Figure 3. (A) HUT102 cell cytotoxicity measured by inhibition of protein synthesis using L3H1leucine incorporation. See text for details of assay. Key: m, ricin in the presence of 60 mM lactose; A, IL2-RTB-RTA in the presence of 60 mM lactose; 0, DAB38gIL2 in the presence of 60 mM lactose. (B) Same as A except OVCAR3 cell cytotoxicity. (C) Same as A except CEM cell cytotoxicity. Standard deviations shown for single assay performed in triplicate. Standard deviation bars < 2% not shown. Each assay performed on three separate occasions, and standard deviations of IDso's shown in Table 1.
structure of ricin cocrystallized with lactose showed lectin sites in the la and 2y subdomains remote from the N-terminus of RTB (21). Their independent behavior from the ligand will permit the systematic modification of sugar binding in recombinant ricin fusions. Previous work by Youle, Goldmacher, and Newton suggested some
112-Ricin Fusion Toxin
residual lectin activity was critical for RTB enhancement of targeted ricin immunotoxin cytotoxicity ( 4 , 3 2 ,33). Since the K, of RTA-RTB association is M-l and our experiments employed concentrations of about M, we expected 50%of IL2-RTB to reassociate with RTA (26). Our observation of 20% heterodimer formation suggests moderate efficiency of reassociation. Again, the X-ray structure shows the RTB amino acid residues interacting with RTA include RTB Al, D2, C4, F140, V141, F218, K219, N220, P260, and F262 (22). Thus, several residues are near the N-terminus and may affect reassociation, but the majority are distant from the surface N-terminal region. The recombinant heterodimers were stable a t high dilutions, suggesting disulfide bond formation between RTA C259 and the free thiol of RTB C4 of IL2-RTB. The IL2-RTB molecule alone was nontoxic to cells with or without the IL2 receptor and with or without lactose (see Results). Thus, no membrane lytic or other toxophore functions have been detected on the RTB moiety either alone or when targeted to cell surfaces with an alternate ligand. In contrast, IL2-RTB-RTA showed cytotoxicity in the absence of lactose indistinquishable from ricin. Thus, all the intoxication functions of ricin were present on the IL2-ricin fusion protein. In the presence of lactose, IL2-RTB-RTA showed selective and potent cytotoxicity to cells bearing the high affinity IL2 receptor (HUT102). The level of cytotoxicity to high affinity IL2 receptor containing cells was on the same order of magnitude as DAB389IL2, a diphtheria toxinIL2 fusion currently in clinical trials, and suggested the ricin-based genetically engineered immunotoxin was potent. Cells containing low levels of intermediate affinity receptor (CEM) or no receptor (OVCAR3) were no more sensitive to IL2-RTB-RTA than ricin alone in the presence of lactose. Future work will be focused on the partial removal of lectin function in the fusion toxin by site-specific mutagenesis. Such fusion toxins lacking significant normal tissue binding could then be studied in animal models of leukemia, lymphoma, or autoimmune diseases. We have prepared single-site and double-site RTB mutants. To date, significant residual cell binding and heterodimer cytotoxicity has been observed even with double-site mutants, suggesting three or more lectin sites on RTB (unpublished observations). Competition experiments with excess ligand have been a frequently used method for confirming specificity of ligand-toxins (14,15). Initial efforts to block our IL2toxins with excess IL2 resulted in complete blockage of DAB3891L2cytotoxicity but minimal reductions in IL2RTB-RTA killing. We reasoned that the residual lectin binding of the chimeric protein may have been strengthened after interaction with the cell surface and that the multivalent binding accelerated internalization a t 37 "C. When we reduced the temperature to 4 "C to impair internalization and shortened exposure time to 30 min, we were able to block cytotoxicity with IL2 plus lactose. Thus, we were able to confirm the extreme rapidity of ricin fusion protein intoxication of cells and establish IL2 receptor specificity of the molecule. This report describes successful production of a ricin toxin fusion protein with full biological potency. The reagent was produced in sufficient amounts and purity for studies of cell intoxication pathways. In the future, modified forms of this fusion protein (with reduced RTB lectin character) may be useful for in vitro and in vivo purging of IL2 receptor bearing cells.
Bioconjugate Chem., Vol. 6,No. 6,1995 671 ACKNOWLEDGMENT
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