Bloconlugate Chem. 1003, 4, 63-68
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Affinity Purification and Characterization of Anti-Tac(Fv)-C3-PE38KDEL: A Highly Potent Cytotoxic Agent Specific to Cells Bearing IL-2 Receptors Cheryl Spence,t Michele Nachman,t Maurice K. Gately,t Robert J. Kreitman,$ Ira Pastan,#and Pascal Bailon'st Protein Biochemistry and Immunopharmacology Departments, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110, and Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892. Received August 14, 1992
A chimeric, single chain antibody fused immunotoxin, denoted anti-Tac(Fv)-C3-PE38KDEL, was engineered and expressed in Escherichia coli. The microbially expressed anti-Tac(Fv)-C3-PE38KDEL was solubilized from inclusion bodies using guanidine hydrochloride, and subsequently refolded in a redox buffer via thiol/disulfide exchange. The recombinant immunotoxin from the crude extract was purified employing receptor-affinity chromatography, which is based upon biological function and involved the immobilized p55 subunit of human IL-2 receptor. The cytotoxic activity of this immunotoxin was measured by the IL-2 dependent phytohemagglutinin (PHA) blast proliferation inhibition and HUT-102 protein synthesis inhibition assays, in which the IC60 values were 41.5 and 0.8 pM, respectively. The biochemical homogeneity and authenticity of the purified material were determined by gel permeation chromatography, amino acid composition and N-terminal sequence analyses, SDS-PAGE, isoelectric focusing, and Western blotting. The receptor-affinity-purified immunotoxin was shown to be highly is a powerful effective in specifically killing cells bearing IL-2 receptors. Anti-Tac(Fv)-C3-PE38KDEL immunosuppressant which may be a potentially useful therapeutic agent in the prevention of allograft rejection and in the treatment of autoimmune diseases. Another anticipated application of this fusion protein is as a chemotoxin in the treatment of some forms of cancer.
INTRODUCTION
Immunotoxins are cytotoxic agents that are designed to selectively kill cells bearing targeted antigens or receptors. The potential clinical applications of immunotoxins include their use as immunosuppressants in the prevention of allograft rejection and in the treatment of autoimmune diseases (1-3). Another emerging application of immunotoxins is their use as chemotherapeutic agents in the treatment of cancer, AIDS, and immunological disorders (4, 5). Recombinant DNA technology has allowed the production of chimeric toxins consisting of growth factors, lymphokines, or antibodies linked to modified diphtheria toxin, Pseudomonas exotoxin (PE), or other toxins. Recently, a hybrid immunotoxin, denoted anti-Tac(Fv)PE40, has been constructed (6). This immunotoxin consists of peptide-linked variable domains of the heavy and light chains of anti-Tac [monoclonal antibody to the low-affinity p55 subunit of human interleukin-2 (IL-2) receptor] fused to a truncated form of Pseudomonas exotoxin, which lacks the binding domain. To increase cytoplasmic delivery and hence toxin activity, anti-Tac(Fv)-PE40 has been further modified to include the characteristic endoplasmic reticulum retention sequence, KDEL, in place of the C-terminal sequence, REDLK, in PE40 (7).Deletion of amino acids 365-380 in PE40KDEL, which removes a disulfide bond but does not decrease activity (81, yielded anti-Tac(FvI-PE38KDEL. The effects of these and other changes in anti-Tac(Fv)-PE40 were
* Author to whom correspondence should be addressed. t Protein Biochemistry Department, Hoffmann-LaRoche, Inc. f
Immunopharmacology Department, Hoffmann-La Roche,
Inc. 5
National Institutes of Health. 1043-1802/93/2904-0083$04.oo/o
recently studied (9). Finally an additional peptide connector, C3 (ASGGPE), is used to connect the light chain (Fv)to PE, resulting in anti-Tac(Fv)-C3-PE38KDEL. This connector resulted in improved renaturation yields in B3(Fv)-PE38KDEL (10). This recombinant immunotoxin is a 62.7-kDa protein containing 587 amino acids. Here, we report the purification of this immunotoxin employing receptor-affinity chromatography (RAC), as well as the biochemical characterization of the purified molecule. EXPERIMENTAL PROCEDURES
Plasmid Construction and Protein Expression, Plasmids encoding anti-Tac(Fv)-C3-PE38KDEL were expressed after transforming them into BL21 (XDE3) E. coli, as described (9-1 3). The cultures of E. coli were grown in a 10-L fermentor at 37 "C to an A650 of 6-8 and induced with isopropyl 8-D-thiogalactoside to a final concentration of 1 mM, for 90 min at 37 OC. Bioassays. Protein Synthesis Inhibition Assay. Cytotoxic activity of the purified immunotoxin was determined by its inhibition of protein synthesis in HUT-102 cells as measured by the [3Hlleucine incorporation as described (12). IL-2 Dependent PHA Blast Proliferation Inhibition Assay. In this assay, cytotoxicity is measured by the decrease in thymidine incorporation into cellular DNA as described in detail elsewhere (12). A reference anti-Tac(Fv)-C3-PE38KDEL,which has been assigned to activity of 400 unit//zg,was used as a standard. Using this standard, 1uniVmL of anti-Tac(Fv)-C3-PE38KDEL routinely caused approximately 50% inhibition in the assay. Preparation of Receptor-Affinity Adsorbent. The production and purification procedures of a soluble form of human IL-2 receptor (~551, denoted IL-2RANae, have 0 1993 American Chemical Society
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Bioconlugete Chem., Vol. 4, No. 1, 1993
been described elsewhere (14). The purified IL-2R was immobilized on NuGel-Polyaldehyde (500 A, 40-60 pm, Separation Industries; Metuchen, NJ) according to the following procedure. Fifty grams (100 mL) of dry gel was washed three times in a sintered-glass funnel with 100mL of ice-cold water. The gel was transferred to a stoppered 500-mL Erlenmeyer flask, containing 100 mL of a 2.0 mg/ mL IL-2R solution in phosphate-buffered saline (PBS, pH 7.4). Six hundred milligrams of sodium cyanoborohydride was added to the gel suspension to a final concentration 0.05 M. The mixture was shaken for 16 h at 4 "C. The uncoupled IL-2R was collected by filtration. The gel was washed twice with 100mL of PBS. The filtrate and washes were pooled and a small aliquot was dialyzed against PBS. The gel was then treated for 2 h with 100 mL of 1.0 M ethanolamine, pH 7.0, in the presence of 0.05 M sodium cyanoborohydride. It was washed with PBS and stored in 0.02% sodium azide at 4 "C. The volume of the uncoupled protein solution was noted and the protein content was determined from its UV absorbance at 280 nm, using the A2w value of 1.65 for a 1 mg/mL IL-2R solution. The difference between the amount of IL-2R present in the starting reaction mixture and the amount remaining after coupling, divided by the volume of the gel (100 mL), gave the IL-2R coupling density (mg/mL of gel). Solubilization and Renaturation of Anti-Tac(Fv)C3-PE38KDELfrom E. coli. All operations were carried out at 2-8 "C unless otherwise noted. The frozen E. coli cell paste was washed twice in 4 volumes (4 mL/g of cells) of 100 mM Tris-HC1, pH 8.0, containing 5 mM EDTA, and during the washings the pH was adjusted to 8.0, if necessary. The supernatant and pellet were collected by centrifugation at 17000g for 20 min. The pellet was uniformly suspended in 20 mM Tris-HC1, pH 8.0, containing 0.2% sarkosyl and 1.5 mM EDTA. The supernatant and pellet were collected as before. The pellet was suspended in 5 volumes (5 mL/g of cells) of 6 M guanidine hydrochloride (Gu-HC1)in 100 mM Tris-HC1, pH 8.0,1.5 mM EDTA, 1 mM reduced glutathione (GSH), and 0.1 mM oxidized glutathione (GSSG). The suspension was pulse-sonicated six times for 30 s, a t 50% power, with a Vibra Cell Sonicator (Sonicsand Materials, Inc.; Danbury, CT). The homogenate was stirred for 60 min at room temperature. The supernatant containing the solubilized anti-Tac(Fv)-C3-PE38KDEL was collected by centrifugation at 31000g for 30 min. It was then rapidly added to 19 volumes (20-fold dilution) of a vigorously stirring 20 mM Tris-HC1, pH 8.0 buffer, containing EDTA, GSH, and GSSG (redox buffer) at concentrations as described before. The pH was maintained at 8.0. The diluted extract was stirred for 60 min and allowed to stand for 3-4 days. The extract was clarified by centrifugation at 17000g, followed by filtration through a0.8/0.2 pm filter (Sartorius; Yauco, Puerto Rico). This was the starting material used for the receptor-affinity purification of anti-Tac(FvbC3PE38KDEL. Receptor-AffinityChromatography. One hundred milliliters of immobilized IL-2R gel were packed into an Amicon G-44 X 250 column fitted with two adapters. The column was equilibrated with PBS buffer and 20 L of the clarified extract (obtained from 200 g of cells) was applied to the column at a flow rate of 10 mL/min. The column effluents were monitored by a Gilson l l l b UV detector and recorded by a Kipp and Zonen recorder. The column was washed with PBS until the UV absorbance at 280 nm returned to b'ase-line. The specificallyadsorbed anti-Tac(Fv)-C~-PE~~KD was E Lthen eluted from the receptor
column with 3 M potassium thiocyanate in PBS buffer. The eluate was dialyzed against PBS buffer. Gel Permeation Chromatography. A Pharmacia K501100 cm column was packed with SephacrylS-200 HR (Pharmacia LKB Biotechnology; Piscataway, NJ) to a height of 95 cm. The column was equilibrated with PBS buffer at a flow rate of 0.85 mL/min and calibrated with standard high and low molecular weight marker proteins (Pharmacia LKB Biotechnology). The concentrated antiTac(Fv)-C3-PE38KDEL(40-50 mL) was applied onto the column and developed with the equilibration buffer. The column operations were monitored as described in the affinity purification step. Four-minute fractions were collected using the LKB Ultra Rac-7000 fraction collector. The peak corresponding to the monomer (-63 kDa) was collected, filtered through a 0.2 pm filter, and stored at 4 "C. Protein Determination. Protein content was determined by the Bradford method (15) using bovine serum albumin as the standard, and the values were confirmed by quantitative amino acid analysis (16). SDS-PAGE Analysis, Isoelectric Focusing, and Western Blotting. The immunotoxin from various stages of the purification scheme was analyzed by sodium dodecyl (lauryl) sulfatelpolyacrylamide (12 % ) gel electrophoresis (SDS-PAGE) under nonreducing conditions according to the methods of Laemmli (17). The isoelectric focusing of anti-Tac(Fv)-C3-PE38KDEL was performed with LKB's isoelectric focusing unit, using 3-10 p1 range ampholytes according to the manufacturer's instructions. Western blotting of the purified immunotoxin at concentrations ranging from 0.05 to 0.2 pg was carried out by transferring anti-Tac(Fv)-C3-PE38KDEL to nitrocellulose paper following SDS-PAGE, according to standard procedures. In two separate experiments, blotting was performed with antibodies against murine IgG (H and L chains) and with antibody to Pseudomonas exotoxin A. The respective detection antibodies were conjugated with horseradish peroxidase. Amino Acid Analysis. Amino acid analyses were performed with a postcolumn fluorescamine amino acid analzyer (16). Samples were hydrolyzed in 6 N HC1 containing 4% thioglycolic acid at 110 "C for 20-24 h in vacuo. RESULTS
Solubilization of Immunotoxin from Inclusion Bodies. Prior to disrupting the transfected E. coli cells by sonication to release the inclusionbodies,the cells were washed sequentially in Tris and sarkosyl buffers. These initial washes removed significant d o u n t s of unwanted soluble proteins from the cell pellet (Figure 1, lanes 1-3). When the GwHC1 extract was analyzed by SDS-PAGE, a prominent, -63-kDa band was observed, which corresponded to the expected molecular weight of the immunotoxin (Figure 1, Lane 4). Receptor-Affinity Purification. A soluble form of the low-affinity p55 subunit of IL-2R was chemically coupled to silica-based NuGel-Polyaldehyde via reductive amination, at a coupling density of 1.35 mg/mL gel. The coupling efficiency was 40%. The affinity sorbent's binding capacity for anti-Tac(Fv)-C3-PE38KDEL was experimentally determined as described (18) and found to be 1.65 mg/mL of gel, which translates into a binding efficiency of 47 5%. The receptor affinity column (100mL) used in this study had an immunotoxin binding capacity of 165 mg. Immunotoxin was purified from dilute, crude extract employing receptor-affinity chromatography (RAC, 18).
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Purification of 11-2 Receptor Specific Immunotoxin S
1
2
3
4
kDa 97.4 66.:
-63kDa
45
The experimentally determined N-terminal sequence of the immunotoxin monomer was identical to the unique sequence of the encoded immunotoxin (data not shown). In addition, all molecules contained N-terminal methionine. Finally, the amino acid composition results (Table 111) agreed well with the expected values.
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DISCUSSION
21.!
Anti-Tac(Fv)-C3-PE38KDEL is expressed in E. coli as an insoluble form within the inclusion bodies, which necessitates the use of a strong denaturant such as 6 M GwHC1 for its solubilization. Sonication in the presence of 6 M GwHC1 not only disrupts the cell wall and membrane, resulting in the release of the inclusion bodies, but also helps to solubilize the immunotoxin (Figure 1, lane 4). Since the Gu. HC1-solubilized immunotoxin has been denatured, it must be renatured. This requires either a substantial reduction in the concentration of GwHC1 or its complete removal. It has been shown previously that optimal protein refoldingoccursat or below the micromolar range (20). For this purpose and for the reduction of GwHC1 concentration, the extract was diluted 20-fold. A redox buffer was chosen for dilution, because previous studies have indicated that S-S bond formation (i.e., protein folding)could be initiated and maintained via thiol/ disulfideexchangebetween the naturally occurringprotein sulfhydryl groups and a redox buffer environment (21). The receptor-affinity purification of anti-Tac(Fv)-C3PE38KDEL is based upon the specific and reversible interaction between the immobilized IL-2R (Tac) and the freely available anti-Tac in the fusion protein. The receptor-affinitysorbent is capable of preferentially binding correctly folded monomers from a heterogeneous population of molecules (22). Hence, in the RAC purified immunotoxin, at least the anti-Tac(Fv) portion is expected to be completely renatured. Gel permeation chromatography is essential for the separation of the desired monomeric form of immunotoxin from the inactive high molecular weight aggregates. It should be pointed out that GPC under aseptic conditions is an ideal tool for the exchange of biotherapeutics such as anti-Tac(Fv)-C3-PE38KDEL into the final storage or formulation buffer. It also serves the purpose of removing trace contaminants such as endotoxins, E. coli proteins, degradation products, and salts,among others. In general, GPC is an integral part of any purification scheme used for the preparation of preclinical and clinical-gradeprotein therapeutics. The apparent discrepancy between the appearance of high molecular weight peaks in the GPC profile (Figure 3) and their obvious monomeric content as seen on SDSPAGE (Figure 4) suggests that some dissociation of the aggregates may have occurred upon their exposure to SDS. The receptor-affiiity adsorbent's demonstrated preference for binding correctly folded monomers (22) lead us to believe that the RAC-purified material was originally monomeric. The high molecular weight aggregates observed during GPC might have been formed during dialysis and concentration,before applicationonto the gel filtration column. The bioactivities of the extract and RAC flowthrough materials (Table I) could not be reliably determined in the IL-2 dependent PHA blast proliferation inhibition assay due to their extremely dilute nature as well as the possible presence of interfering substances. Regardless, since the amount of immunotoxin (146 mg) passed through the column did not exceed the total binding capacity (160 mg) of the affinity column, no activity was expected to
14.4
Figure 1. SDS-PAGE analysis of E. coli cell washings and GwHC1 extract under nonreducing conditions: lane S, standard molecular weight marker proteins; lane 1, Tris wash 1; lane 2, Tris wash 2; lane 3, Sarkosyl wash; lane 4, GwHC1 extract (neat).
The protein contents and activities of immunotoxin obtained from 200 g E. coli cell paste at various stages of the purification scheme are listed in Table I. The immunotoxin recoveries were 146 mg (100%) after RAC. Protein recoveries after GPC were 31 mg (21%), 9 mg (2%),and 57 mg (39%),respectively, for GPC peaks 1,2, and 3. The GPC protein values account only for the major portion of the peaks and exclude the overlapping parts (see Figure 3), thereby reducing the percentage of the protein recoveries. The SDS-PAGE profile of the RACpurified material indicated a major band around 63 kDa (Figure 2, lane 3). However, upon gel permeation chromatography (GPC), the RAC-purified material yielded three peaks (Figure 3), whose SDS-PAGE profiles under nonreducing conditions are shown in Figure 4. Peaks 1 and 2 corresponded to high molecular weight aggregates, >400 and -250 kDa (determined from molecular weight standard curves),respectively,yet all three peaks contained the -63-kDa band expected for the monomeric protein. An IL-2 dependent PHA blast proliferation inhibition assay was used to determine the bioactivities of the immunotoxin at various stages of the purification scheme (Table I). The 18.1 X lo6 units of activity found in the RAC eluate, with a specificactivity of 1.26 X lo5units/mg, was taken as 100% . The specific activities of GPC peaks 1,2, and 3 were 0.26 X lo5, 2.54 X lo5, and 4.0 X lo5units/ mg, respectively. The recovery data indicates that the total activity present in the final product (monomer) is 126% of the activity in the starting material (RAC eluate). In Table 11, the bioactivity of anti-Tac(Fv)-C3-PE38KDEL is compared with that of anti-Tac(Fv)-C3-PE40 (the parent immunotoxin with the C3 linker) and IL2PE664G1u (the most cytotoxic IL2-PE construct to date, 19). In both assays, anti-Tac(Fv)-C3-PE38KDEL was the most active immunotoxin construct, with IC50values of 41.5 and 0.8 pM for the PHA and HUT-102 assays, respectively. The anti-Tac(Fv)-C3-PE40 construct had intermediate activity and the IL2-PE664G1u had the least cytotoxic activity. Biochemical Characterization. SDS-PAGE analysis of the monomer under nonreducing conditions indicated a single 63-kDa protein, devoid of any dimers, oligomers, or degradation products (Figure 4, lane 3). Similarly, isoelectric focusing results indicate a major band with a PIof 5.4 (Figure 5). Western blotting with antibodies to murine IgG and PE both show a prominent band at -63 kDa (Figure 6). However, blotting with anti-PE also indicated the presence of two additional minor bands (
