Proc. Nad. Acad. Sci. USA

Vol. 89, pp. 3075-3079, April 1992 Biochemistry

Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: Influence of interdomain connections [B3(Fv)-PE38KDEL/renaturadon/protein engineering]

ULRICH BRINKMANN, JOHANNES BUCHNER*,

AND IRA

PASTAN

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, 37/4E16, Bethesda, MD 20892

Contributed by Ira Pastan, December 26, 1991

ABSTRACT We have studied the refolding of completely unfolded and reduced Pseudomonas exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable region connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncated form of PE. We have found by direct activity assays that different functional domains of these multf onal proteins fold independently with different kinetics. The ADPribosylation domain of PE and of the recombinant immuntoxin fold rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly. The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immuoxins. To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II of PE. These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules. A mutation that decreases aggregation increases the rate of formation and the yield of active immunotoxin molecules.

recognizes an antigen present on many cancer cells (11, 12). B3(Fv)-PE38KDEL is very cytotoxic to B3 antigen containing cancer cell lines and causes complete regression of solid tumors in mice (10). The procedure used to prepare recombinant immunotoxins produces sufficient material for laboratory use, but the yield of molecules that are specifically cytotoxic to target cells and therefore considered to be properly folded is still lower than that achieved for tissue plasminogen activator or for Fab fragments from inclusion bodies (13). Possible reasons that contribute to low yield are that single-chain immunotoxins take a long time to fold into an active state and tend to aggregate during the folding process. Recombinant immunotoxin molecules are artificial structures composed of moieties that can independently fold properly; both the Fv fragment and the toxin part refold alone with good yields (14, 15). Thus, the problem in the renaturation of B3(Fv)-PE38KDEL might be due to the nonproductive interaction of the individual entities. Folding of individual domains in multidomain-multifunctional proteins is a well established process in the acquisition of structure of proteins (16). However, few direct measurements of the activity of separate domains during the folding of multidomain proteins have been described and these are based mostly on the separate folding of proteolytically derived fragments (17-19). PE and the PE-derived single-chain immunotoxins can be analyzed by activity assays to determine whether individual domains fold independently from each other. To obtain the complete function of these complex molecules, which is the ability to kill target cells, proper folding of all domains is essential. Conversely, the ADPribosylation activity of domain III can be assayed independently from the function of the other domains. Here we show that different functional entities of PE and of several variants of a single-chain immunotoxin fold with different kinetics, indicating that they fold independently from each other. We further demonstrate that variations in the peptide connecting the antibody domain to the toxin influence the folding kinetics, the tendency to aggregate, and the yield of properly folded molecules.

Pseudomonas exotoxin (PE) is a 66-kDa multifunctional protein that kills eukaryotic cells by ADP ribosylating elongation factor 2 (1-3). PE consists of three major structural domains, each having at least one distinct function. Domain Ia is responsible for cell binding, and domain II mediates translocation of the ADP-ribosylating portion (domain III) of PE into the cytosol of the cell (4). In addition, a small sequence at the carboxyl end of PE delivers the molecule to an intracellular compartment from which translocation occurs (5, 6). Deletion or inactivation of any of these domains results in loss of cytotoxicity. It is possible to substitute for the function of the cell binding domain by replacing domain Ia with other cell binding molecules, including antibody variable domains (Fv) connected by an artificial peptide linker. The resulting recombinant immunotoxins bind to and selectively kill cells that are recognized by the antibody (7-10). These recombinant toxins are produced in Escherichia coli, where they accumulate as cytoplasmic inclusion bodies (IBs). lBs are easy to purify and often contain almost pure recombinant protein, which can be refolded to active protein. Recently, we described the production of a single-chain immunotoxin, B3(Fv)-PE40, and a derivative with an altered carboxyl terminus and an internal deletion of 15 amino acids that has enhanced cytotoxic activity, B3(Fv)-PE38KDEL (10). These molecules have the cell binding domain of PE replaced by the Fv region of the monoclonal antibody B3 (see Fig. 1), which

MATERIALS AND METHODS Construction of Single-Chain Immunotoxin Variants.

pULI1 (10) carries the gene for the single-chain immunotoxin B3(Fv)-PE40 under control of the T7 promoter. The connection of the Fv coding portion to PE40 has a HindIII recognition site and encodes the amino acid sequence KAFGG (from plasmid pVC38H; ref. 9; Fig. 1). Two derivatives ofthis sequence were made by PCR (22) with primers containing a HindIII recognition sequence, P2 (5'-AACATAAAGCTTGAbbreviations: PE, Pseudomonas exotoxin; IB, inclusion body. *Present address: Institut fuer Biophysik und Physikalische Biochemie, Universitaet Regensburg, Universitaetsstrasse 31, D-8400 Regensburg, Federal Republic of Germany.

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3075

3076

Biochemistry: Brinkmann et al.

Proc. Natl. Acad. Sci. USA 89 (1992) FIG. 1. Features of different variants

b

a PE

B3(Fv)-PE40

B3(Fv)-PE38KDEL cellbinding

ADPlocation ribosylation

trans-

NH2 -MANLAEEAF GSL.. ..IKPVISHRLHFPE GGSL..

GTTGGCCGAAGAGGCTTICAA-3') and P3 (5'-AACATAAAGCTTCCGGAGGTCCCGAGGGCGGCAGCCTG-3'), to introduce mutations and another primer (5'-GGCTTT1-l GTTAGCAGCCGAA-3') complementary to a region immediately 3' to the PE40 gene close to a unique EcoRI site (10). As template to obtain the mutant C2, we used the PE40 coding region of pJY8SL (23) because this connection sequence is similar to the amino terminus of LysPE40 encoded by that plasmid (the amino terminus of LysPE40 is MANLAEEAFK, C2 is KAWLAEEAFK). The template to- make the C3 variant was pVC45F+T (21). PCR was performed with an annealing temperature of 450C using 10% formamide. PCR fragments were separated from primers by Centricon 100 concentrators (Amicon) and cleaved with HindIII and EcoRI to replace the PE40 coding region in pULIL. Mutant clones were confirmed by DNA sequencing. B3(Fv)-PE38KDEL derivatives were constructed from the B3(Fv)-PE40 variants, replacing a Sal I/EcoRI fragment by the corresponding fragment of pULI3 (10), which contains a deletion in the PE40 gene from amino acids 365-380 (of PE) and encodes KDEL at the carboxyl terminus (5). Production of Recombinant Immunotoxins. Expression plasmids were transformed into E. coli BL 21 (ADE3) (24) and grown in superbroth containing 2% glucose, 0.05% MgSO4, and 100 jig of ampicillin per ml and induced at an OD600 of 3 with isopropyl 83-D-thiogalactopyranoside (final concentration, 1 mM) for 2 hr. Outer membrane and periplasmic proteins were removed from the cells by osmotic shock, the spheroplast pellets were resuspended in 50 mM Tris HCI/20 mM EDTA, pH 7.4 (TE50/20), and lysozyme was added to a final concentration of 200 pg/ml. After a 1-hr incubation at 20'C, NaCl and Triton X-100 were added to final concentrations of 0.5 M and 2%, respectively, and IBs were sedimented by centrifugation after an additional 30-min incubation. The IBs were resuspended in TE50/20 and washed four times and stored as pellets at -70°C. Refolding from IBs and Purification. IBs or lyophilized PE protein were denatured and reduced in 6 M guanidine hydrochloride/0.1 M Tris.HCl/2 mM EDTA/0.3 M dithioerythritol, pH 8.0, for 2 hr and diluted in the same buffer to 5 mg/ml. Refolding was started by rapid 1:100 dilution into 100 mM Tris-HCI/500 mM L-arginine/2 mM EDTA/8 mM glutathione disulfide, pH 8. Unless otherwise noted, refolding was performed at 10°C. For varying parameters and to obtain kinetic data, aliquots of the refolding solution were passed through a Centricon 100 concentrator to remove aggregates and after addition of human serum albumin (HSA) to 0.2%, the solution

of B3(Fv) immunotoxins. (a) Single-chain

immunotoxins are composed of the Fv domains of a monoclonal antibody connected to truncated forms of PE (PE40). The carboxyl end of the heavy-chain variable region (VH) is connected by a (Gly4K Ser)3 linker to the light-chain variable reE gion (VL), which is then fused to PE40. L The toxin part includes, in addition to the translocation and ADP-ribosylation domain, a small domain (Ib; see ref. 20) with unknown function that is not essential for activity. B3(Fv)-PE38KDEL lacks this doC1 main and has a carboxyl-terminal sequence KDEL to enhance its activity (6). C3 (b) Variants of B3(Fv)-PE38KDEL differ only in the region connecting the Fv doC2 mains to the toxin. The connector C1 is present in immunotoxins that are derivaof pVC38H (9). Connector C2 is PE40 tives similar to the amino terminus of Lys PE40 PE (21) and C3 resembles the PE sequence immediately preceding domain II.

was immediately frozen in dry ice. Other aliquots were frozen directly. For purification of immunotoxins, we dialyzed the refolding solution at 4°C against 20 mM Tris-HCl/100 mM urea, pH 7.4, and separated properly folded proteins from aggregates and contaminating proteins by Q-Sepharose, Mono Q, and TSK250 chromatography as described (10). Protein concentrations were determined by the method of Bradford (25). Cytotoxicity Assays. A431 cells and L929 mouse fibroblasts were incubated for 24 hr in 96-well plates containing 1.6 x 104 cells in 200 ,ul of medium each, with various concentrations of refolding solution. Inhibition of protein biosynthesis was determined by measuring the incorporation of [3H]leucine into the cell proteins as described (10). To determine the yield of correctly folded molecules at different time points after the start of renaturation, we incubated the target cells with dilutions of the refolding solution in phosphate-buffered saline (PBS)/0.2% bovine serum albumin. The cytotoxic assay includes a 24-hr incubation of the cells with samples in which unfolded or partially folded proteins may continue to fold into active molecules, thus influencing the assay. However, in the case of single-chain immunotoxins, the time period in which this might influence the result is 1 hr or less after renaturation is initiated, because those molecules are rapidly degraded in the cytotoxic assay with a t1/2 of -20 min (data not shown). Properly folded PE is more stable (t1/2 4 hr); thus, refolding during the assay theoretically could influence the results, but partially or completely unfolded PE should not be stable in the cytotoxicity assay. ADP Ribosylation of Eukaryotic lion Factor 2. ADPribosylation assays using wheat germ extracts were carried out as described (26). For determination of the ADP-ribosylation activity of refolded PE, we incubated samples for 15 min at 20°C in 4 M urea/40 mM dithiothreitol to activate the molecules before the assay. Recombinant immunotoxins do not need this activation, like other PE derivatives that have the domain I partially unfolded (27) ordeleted (23). Samples were diluted 1:20 in PBS/0.2% bovine serum albumin directly before the assay. The overall incubation period in this assay is 10 min for immunotoxins and 25 min for PE. Because we cannot exclude that some refolding takes place in this time period, the data obtained at very early time points might show higher activities than were actually present in the sample.

RESULTS Functional Subunits of Denatured and Reduced PE and Single-Chain Immunotoxins Regain Function with Different

Biochemistry: Brinkmann et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 2. Reactivation kinetics of PE and B3(Fv)-PE38KDEL. The yield of correctly folded molecules in the renaturation solution at different time points after the start of renaturation was determined by cytotoxicity assays (see Materials and Methods) using A431 cells for immunotoxins and L929 cells for PE. The refolding solution without protein did not interfere with the assays. The relative amount of correctly folded protein was calculated by setting the highest value of cytotoxic activity to 100%o so the other activities are relative to this. ADP-ribosylation activities were determined as described.

total cell protein. After renaturation, we purified active monomeric proteins to homogeneity and analyzed them for their ability to kill A431 cells. Fig. 3a shows that all variants have the same cytotoxic activities. We also analyzed the stability of the molecules in various buffers and were unable to detect any differences (Fig. 3b). We conclude that the mutations introduced do not affect the function and stability of the protein. We noticed, however, a significant difference in the yields of properly folded molecules. As shown in Fig. 4, the immunotoxin with C3 gives the highest yield of correctly folded molecules, whereas the C2 sequence causes a reduction in the yield. This difference in yield is even more dramatic where renaturation is carried out at 200C. The Connection Between the Fv and the Dom II of PE Affects Folding Kinetics and Propensity to Aggregation of Single-Chain- Immunotoxins. To investigate possible reasons for the differences in refolding yield between "connector variants," we analyzed their folding kinetics. Fig. 5 shows that the immunotoxin with C3 folds faster than molecules containing C1 and in greater yield. The C3-containing protein reached maximal activity levels after -20 hr, whereas it took 40 hr for the molecules containing C1 and even longer with C2. The differences in kinetics were observed when assaying the complete function of the molecules by their ability to kill cells. However, the kinetics of appearance of ADPribosylation activity was similar with all three molecules with maximal activity achieved within 1 hr. This result confirms that the separate functional parts regain their active conformation independently. Variations in the connector also influence the aggregation of these molecules during the initial process of refolding. This aggregation occurs immediately after diluting the solubilized proteins into the folding buffer and was monitored by light scattering (29). As shown in Fig. 6, the variant with the C3 connector aggregated less. This variant folds faster and with better yield than the other variants. The other two molecules, with the C1 and C2 connector, show the same tendency to aggregate, which is greater than that of the C3-containing molecule. Aggregation can be influenced by the addition of

Kinetics. While optimizing the folding conditions for B3(Fv)PE38KDEL, we observed that the protein folds slowly. The maximum yield of properly folded molecules is reached after >40 hr (Fig. 2b). This rate is unusually slow when compared to other complex multidomain molecules (13). We examined the kinetics of reactivation ofreduced and unfolded PE under the same refolding conditions and found that PE renatures much faster, with complete renaturation achieved in 2.5 hr (Fig. 2). We also measured the kinetics of- refolding of the ADP-ribosylation domain of PE and found that ADPribosylation activity is fully restored in