INFECTION
AND
IMMUNITY, Jan. 1991, p. 407-414
Vol. 59, No. 1
0019-9567/91/010407-08$02.00/0 Copyright C 1991, American Society for Microbiology
Analysis of Pseudomonas Exotoxin Activation and Conformational Changes by Using Monoclonal Antibodies as Probes MASATO OGATA, IRA PASTAN,
AND
DAVID FITZGERALD*
Laboratory of Molecular Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892 Received 31 July 1990/Accepted 9 October 1990
Pseudomonas exotoxin (PE) is a protein toxin composed of three structural domains. In its native form, the toxin is a 66,000-M, proenzyme that must be activated to express full ADP.ribosylating activity. To study the process of activation and accompanying conformational changes, we have isolated 10 monoclonal antibodies to a 40,000-Mr fragment of the toxin (PE40) that exhibits full enzyme activity but lacks the toxin's cell-binding domain and contains amino acids 253 to 613 (comprising domains II, Ib, and E). By using mutant PE molecules in which all of domain I and portions of domains II, Ib, and III were deleted, the locations of the epitopes for each of the antibodies were determined. Eight of these monoclonal antibodies were further characterized. Of these eight, all reacted with soluble PE40 and an interleukin-2-PE40 conjugate, but only two reacted strongly with native soluble PE. However, all eight reacted with PE after it had been immobilized on nitrocellulose or after it had been activated to express full ADP-ribosylating activity. Antibodies were also assessed for their ability to neutralize the cytotoxic activity of either PE or interleukin-2-PE40. These antibodies should be useful as probes for monitoring the activation and processing of PE that occur during endocytosis and in determining the location of epitopes that are important for toxin activity.
Pseudomonas exotoxin (PE) is made and secreted by Pseudomonas aeruginosa and may contribute to the pathogenicity of certain clinical isolates by acting as a virulence factor (26). When present at concentrations of 1 to 10 ng/ml, PE is capable of killing mammalian cells, which it does by gaining entry to the cell cytoplasm and catalyzing the ADP ribosylation of elongation factor 2. Recent advances in the study of this toxin include the cloning and expression of the structural gene in Escherichia coli (9) and the analysis of its crystal structure by X-ray diffraction (1). The crystallography data revealed that PE is composed of three prominent structural domains (1). Recent work has focused on defining the role that each of the structural domains plays in the cytotoxic activity of the toxin (10, 25). PE binds and enters cells by receptor-mediated endocytosis and then translocates to the cell cytoplasm, where it inactivates elongation factor 2 and thereby shuts down protein synthesis (7, 12, 23). The amino-terminal domain, called domain Ta, binds to PE receptors on the surface of mammalian cells; then, after internalization via coated pits and endocytic vesicles, domain II mediates translocation to the cytoplasm; finally, the C-terminal domain, domain III, containing the ADP-ribosylating activity, inactivates protein synthesis. In addition to studies focused on toxin structure and function, PE-derived proteins have been used to make immunotoxins and other cytotoxic conjugates for the treatment of cancer and various other human diseases (8, 23). One immunotoxin, anti-Tac-PE, has been given to individuals with adult T-cell leukemia, and another, OVB3-PE, has been given to women with ovarian cancer (unpublished data). PE conjugates have also been developed for vaccine use (3, 6). From the latter studies and from our own experience in phase I clinical trials, it is clear that PE is an excellent immunogen. *
Here we describe the development of several murine monoclonal antibodies that were obtained after immunization with a truncated form of PE, termed PE40. PE40 lacks the toxin's N-terminal binding domain, is relatively nontoxic, and can be injected into animals without the need for "toxoiding." PE40 has at least one other notable property: unlike native PE, which is a proenzyme and requires treatment with urea and dithiothreitol (DTT) to express full enzyme activity (18), PE40 has full activity without any pretreatment (16). This suggests that the removal of domain I alters both toxin structure and function. When native PE is first added to cells, it is presumably still in its proenzyme state. Later, within cells, PE has to be processed and converted to a fully active form. This active form has been partially characterized and appears to be composed of a proteolytic fragment, 37 kDa in size, which is derived from the C-terminus of PE (21a). Of the eight monoclonal antibodies we have characterized, only two react with soluble native PE. However, all eight react with PE after it has been unfolded. Unlike the situation with PE, all the antibodies react with soluble PE40. Here we show that these antibodies can be used to monitor PE activation and to identify key functional epitopes. MATERIALS AND METHODS
Reagents. Hypoxanthine-aminopterin-thymidine was purchased from GIBCO, Grand Island, N.Y. Biotinylated horse anti-mouse immunoglobulin G (IgG; heavy and light chains), avidin DH, and biotinylated horseradish peroxidase H were purchased from Vector Lab, Inc., Burlingame, Calif. 2,2'Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) was purchased from Boehringer Mannheim Biochemical, Indianapolis, Ind., and O-phenylenediamine was purchased from Sigma, St. Louis, Mo. Protein A- or protein G-Sepharose was purchased from Pharmacia LKB, Piscataway, N.J., or Genex Co., Gaithersburg, Md., respectively. Murine antibody class- or subclass-specific antibodies were purchased
Corresponding author. 407
OGATA ET AL.
408
300 253
1
PE
200
100
n
INFECT. IMMUN.
i
400 365 405
500
600
613
Da 1 IuSt ala asn leu
1-3
pVC85 (PE40)
253
pMO235 408
1-3,asp
pVC7
0
1-2
pVC9 oul
1-4 pVC17
his met pCS7
1-3
264
TGFE
1-
400
FIG. 1. Schematic map of PE and mutants used for the epitope mapping of monoclonal antibodies. The map depicts the different regions of PE present in each molecule. The numbers, including those on the scale bar, represent amino acids, with the first amino acid of mature (processed) native PE numbered +1. TGFa, Transforming growth factor a.
from Southern Biotech, Assoc., Birmingham, Ala. PE was purchased from the Swiss Serum and Vaccine Institute, Bern, Switzerland. Plasmids, bacterial strains, and cell lines. The plasmids encoding various mutant forms of PE are shown in Fig. 1. The construction of the plasmids pVC85, pVC7, pVC9, pVC17, pCS7, and pCSll was described previously (25, 28). The construction of the plasmid encoding PE-His274'276'279, in which the arginines at positions 274, 276, and 279 were changed to histidines, was described elsewhere (14). The plasmid pMO235 encoding the entire domain II of PE was constructed as follows. pVC85 was partially digested with SfiI and then completely digested with PpuMI. A 2.9-kb DNA fragment was separated on a low-melting-point agarose gel, eluted, and ligated to an oligonucleotide duplex (5'-CGGCCAACTAACCCGGGTAAG-3' and 5'-GTCCT TACCCGGGTTAGTTGGCCGCGC-3'). In all the plasmids, the genes are linked to a phage T7 late promoter. For expression of these plasmids, E. coli BL21(XDE3), which contains a T7 polymerase gene fused to a lac operator and promoter, was used. Expression and purification of mutant forms of PE were done as described before (15). For the epitope mapping study of monoclonal antibodies by Western immunoblotting, crude cell extracts containing toxin proteins were also used. Gel electrophoresis and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (17). Gels containing between 10 and 17.5% polyacrylamide were used, depending on the size of the mutant form of PE. For immunoblotting, electrophoresed samples were transferred to nitrocellulose paper and processed with anti-PE antibodies as described previously (25). Isolation of monoclonal antibodies. Female BALB/c mice
(8 weeks old) were injected intraperitoneally (i.p.) with 20 ,ig of PE40 in 0.1 ml of complete Freund's adjuvant. One month later, 10 ,ig of PE40 in incomplete Freund's adjuvant was injected i.p. Two months later, 20 ,ug of PE40 was injected intravenously, and 3 days later, spleen cells (1.7 x 108) from an immunized mouse were fused to P3/x63-Ag 8.654 cells by using polyethylene glycol 4000. Hybridomas M40-1, -2, -4, -5, -6, -7, -8, -9, and -10 were isolated from this fusion.
Alternatively, 2 months after the second i.p. injection, PE40 was injected (5 ,ug intravenously plus 5 ,ug i.p.) for two successive days. Two days after the last injection, spleen cells from an immunized mouse were used for fusion, and hybridoma M40-3 was isolated. After fusion, cells were seeded in 96-well tissue culture plates and selected in culture medium containing hypoxanthine-aminopterin-thymidine. The reactivity of monoclonal antibodies to toxins was screened by a strip comb method described before (21). Briefly, 20 ng of PE40 was directly blotted onto a nitrocellulose filter cut into small strips. Strips were incubated with 2% gelatin in phosphate-buffered saline (PBS) at 37°C for 1 h (blocking) and then incubated with the hybridoma culture supernatant at 37°C for 30 min. After being washed with PBS containing 0.05% Tween 20 (T-PBS) three times, strips were incubated with biotinylated horse anti-mouse IgG (heavy and light chains) at 37°C for 30 min. Strips were then washed three times with T-PBS and incubated with avidin-biotinylated horseradish peroxidase complex at 37°C for 15 min. After being washed four times with PBS, strips were incubated with PBS containing, 3,3'-diaminobenzidine (0.5 mg/ ml) and H202 (1:2,500 dilution of 30% H202 solution). Cells secreting the desired antibodies were cloned by limiting dilution, with normal murine spleen cells used as feeder cells. Ten independent cell lines secreting anti-PE40 antibodies (M40-1 to M40-10) were established. The subtype of each monoclonal antibody was determined by enzymelinked immunosorbent assay (ELISA) with antibodies specific for classes or subclasses of murine immunoglobulins. Production and purification of monoclonal antibodies. Hybridoma cells (2 x 106 to 5 x 106) were injected i.p. into BALB/c mice that had been injected i.p. with 0.5 ml of pristane 10 days previously. After 7 to 11 days, ascites containing monoclonal antibodies were obtained. Monoclonal antibodies were purified from ascites or hybridoma culture supematants by using protein A (M40-4, -5, -8, and -10) or protein G (M40-1, -2, -3, -6, -7, and -9) column affinity chromatography. ELISA and ELISA competition assay. Microtitration plastic plates (Immuolon 1; Dynatech Lab. Inc., Chantilly, Va.) were coated with PE or PE40 (0.5 to 1.0 ,ug/ml in 0.1 M carbonate buffer [pH 9.6]) at 37°C for 1 h or at 4°C overnight. Plates were then blocked by 2% gelatin in PBS at 37°C for 1 h, washed three times with T-PBS, and incubated with anti-PE40 monoclonal antibodies. After being washed three times with T-PBS, monoclonal antibodies bound to the antigen-coated plates were detected with a Vectastain Kit (Vector Lab., Inc., Burlingame, Calif.). Briefly, the plates were incubated with horse anti-mouse IgG (heavy and light chains) at 37°C for 30 min, washed three times with T-PBS, incubated with avidin-biotinylated horseradish peroxidase complex at 37°C for 15 min, and washed six times with T-PBS. Either ABTS or O-phenylenediamine (0.5 to 1 mg/ml with 0.1 M sodium citrate buffer [pH 4.0]) was used as a substrate for peroxidase, and the A405 or A490 was measured, respectively. For an ELISA competition assay, 25 RI of competitors in various buffers containing 0.2% human serum albumin (HSA) was added to each well after the wells had been coated with antigen and blocked with 2% gelatin. Then, an equal volume (25 ,ll) of monoclonal antibody (0.2 to 0.4 p,g/ml) diluted in PBS containing 0.2% HSA (HSA-PBS) was added and incubated for 30 min at 37°C. Antibodies that bound to the coating antigen were detected as described above. For each well, relative absorbance was calculated as follows: relative absorbance (%) = [(A - Ab)/(AC- Ab) x
VOL. 59, 1991
100, where A is the absorbance of each well with competitor, AC is the absorbance of a well to which 25 ,ul of buffer solution alone is added instead of competitor (variation of the AC value was within 20% when different buffers were used), and Ab is the absorbance of a well to which 25 ,u of HSA-PBS was added instead of antibodies. Ab was usually less than 5% of AC when O-phenylenediamine was used and was less than 20% when ABTS was used. Treatment of PE with DTT or urea. The solution containing PE (5 mg/ml) or PE40 (1.5 mg/ml) was mixed with equal volumes of TE buffer (50 mM Tris, 1 mM EDTA [pH 8.0]) containing 80 mM DTT, 8 M urea, or 80 mM DTT plus 8 M urea. After incubation at 4°C for 1 h, samples were diluted more than 125-fold with HSA-PBS and used as competitors in the ELISA competition assay. Our preliminary experiments with ELISA showed that the DTT plus urea solution diluted more than 125-fold did not interfere the reactivity of our monoclonal antibodies (data not shown). Treatment of PE with low pH. PE or PE40 was incubated in MES buffer (5 mM MES [morpholineethanesulfonic acid], 5 mM citric acid, 150 mM NaCl, 0.9 mM CaCl2, and 0.5 mM MgCl2, pH adjusted with 1 M Tris) containing 0.2% HSA at various pH values for 30 min at 37°C. Then the samples were neutralized by an equal volume of 0.2 M sodium phosphate buffer, pH 6.0, and used as a competitor in the ELISA competition assay. The pH of the samples after neutralization was approximately 5.9, and in preliminary assays, it was shown that the epitopes on the toxin remained intact at pH 5.9. Also, the antibodies were fully reactive at pH 5.9. Neutralizing assay. PE at a concentration of 1 ng/ml or interleukin-2 (IL-2)-PE40 at 5 ng/ml was mixed with various amounts of each monoclonal antibody. The final antibody concentrations were 0.2, 1.0, 19, and 100 ,ug/ml. Toxicity for cells was then measured on Swiss 3T3 and HUT-102 cells for PE and IL-2-PE, respectively, as described previously (16).
RESULTS Establishment of murine hybridoma cell lines secreting anti-PE40 monoclonal antibodies. Because PE is highly toxic for mice (50% lethal dose [LD50] = 0.2 jig), it is not possible to inject large amounts of the native toxin as an immunogen. Inactivation of PE by treating it, for example, with glutaraldehyde may destroy important antigenic sites or alter the conformation of the toxin. Also, native PE is a proenzyme and undoubtedly has a different structure than enzymatically active forms. Therefore, we chose to use a 40,000Mr fragment of PE, PE40, as the immunogen (Fig. 1). PE40, which lacks domain Ia, the cell-binding domain of PE, is 250-fold less toxic than PE for mice (10). Also, unlike native PE, it has full ADP ribosylation activity (16). Spleen cells from BALB/c mice immunized with PE40 were used for cell fusion. Antibody-secreting hybridomas were then screened by a strip-comb blotting method (21). Reactivity of culture supernatants was tested by blotting samples onto nitrocellulose filter paper to which PE40 had been bound. After screening and cloning, 10 independent hybridoma cell lines secreting anti-PE40 monoclonal antibodies (M40-1 to M40-10) were established. The isotype of each monoclonal antibody was determined by using isotypespecific antibodies. M40-1, -2, -3, -6, -7, -9, and -10 were IgGl(K). M40-4 and -5 were IgG2a(K). M40-8 was IgG3(K). Location of reactive epitopes. To determine the specific regions of PE40 recognized by each antibody, PE40 and various PE-derived proteins were expressed in E. coli, subjected to SDS-PAGE, and then analyzed by Western
ANTIBODIES TO PSEUDOMONAS EXOTOXIN P!-asrmd,
('armmno acids preseWl)
t2
pVCB5 253 613 Ud
3
_
4
5
6
7
8
9
10
409
kDa -43
_ -
.'-' P4L-
---
-
_ so
_
_
S
1-2. .......
pVC9 309-613, -25.7 DVC17 381- 673'
-
--
pVC7 408 613
oM0235 `253- 364m D o? 7. a iI
_ -
*-
.
a
IV -25.7
_S
-25.7 14.3
FIG. 2. Reactivity of monoclonal antibodies to various mutants of PE on immunoblotting assay. The gene products of pVC85 (PE40), pMO235, pVC7, pVC9, and pVC17 were run on SDS-PAGE and blotted on the nitrocellulose filter, and the reactivity of each monoclonal antibody at 2 ,ug/ml was tested. Lane 1, M40-1; lane 2, M40-2; lane 3, M40-3; lane 4, M40-4; lane 5, M40-5; lane 6, M40-6; lane 7, M40-7; lane 8, M40-8; lane 9, murine IgG (2 jig/ml); lane 10, mouse anti-PE40 serum (500-fold dilution). *, Not done. The arrow shows degraded products of PE40.
blotting (Fig. 2). For this analysis, we selected 8 of the 10 monoclonal antibodies (M40-1 to M40-8) that reacted strongly in the strip-comb assay. The results are summarized in Fig. 2 and 3. All of the antibodies tested reacted strongly with both PE (data not shown) and PE40 on Western blotting (Fig. 2). M40-1, -2, and -3 reacted with domain TI of PE (amino acids [a.a.] 253 to 364), while M40-4, -5, -6, -7, and -8 did not. M40-1, -2, and -3 also reacted with a protein composed of a.a. 264 to 613 (data not shown), indicating that these antibodies reacted with an epitope(s) on the C-terminal side of a.a. 264. M40-1, -2, and -3 did not react with a protein composed of a.a. 309 to 613 that lacked the N-terminal half of domain TI, suggesting that these antibodies reacted with epitopes between residues 264 and 308 of domain TI. However, from these results, it is difficult to determine the exact location of the C-terminal boundary, since it is possible that the epitope(s) extends over a.a. 308 and the deletion of a.a. 308 or a few amino acids on the N-terminal side of a.a. 308 could destroy the epitope(s). In summary, the epitopes reactive with antibodies M40-1, -2, and -3 were mapped between residue 264 and residue 308 (Fig. 3). In addition, we tested a mutant of PE in which the arginines at positions 274, 276, and 279 were replaced by histidines (PE-His274 276'279; data not shown). M40-1 and -2 reacted strongly with this mutant protein while M40-3 did not, suggesting that at least one of these amino acids contributes to the epitope of M40-3. M40-8 reacted strongly with a protein composed of almost all of domain III (a.a. 408 to 613) (Fig. 2). The reactivity of M40-8, which was mapped between residues 408 and 613 (Fig. 3), was not analyzed further. M40-4, -5, -6, and -7 did not react with either domain TI (a.a. 253 to 364) or a protein composed of almost all of domain III (a.a. 408 to 613) (Fig. 2) or a protein made up of a.a. 400 to 613 (data not shown). These results suggested that the epitopes that reacted with these four antibodies were located between residues 365 and 399. M40-6 and -7 reacted strongly with a protein composed of a.a. 381 to 613 that lacked domain II and the N-terminal half of Ib, whereas M40-4 and -5 showed very low or no reactivity with that protein. From these results, the epitopes
OGATA ET AL.
410 9
100
INFECT. IMMUN.
200
300
500
365 405
253
1
400
600
TABLE 1. Relative affinity of monoclonal antibodies for PE after various treatmentsa
613
Relative affinity for PE (%)
PE 264 308
-0O
M40-1 M40-2 M40-3
365 399
0-0 M40-4 M40-5 381 399
M4046 M40-7
613
408 M40-8
FIG. 3. Summary of epitope mapping of monoclonal antibodies. The numbers show the amino acid number on the boundary of each epitope. The boundary was determined by either binding ( 0) or nonbinding (0) of monoclonal antibodies to mutant PE proteins, as described in the text.
of M40-6 and -7 were mapped to the C-terminal half of domain lb (between residues 381 and approximately 399). The epitopes of M40-4 and -5 were mapped in domain Ib (between residue 365 and residue 399). It is unclear whether the epitopes of M40-4 and -5 are on the C- or N-terminal side of a.a. 381 due to their marginal reactivity with a.a. 381 to 613. Characterization of antibody binding to PE and PE40 by ELISA. Next, we tested the ability of these antibodies to bind native PE or PE40. Although all antibodies reacted well with both PE and PE40 on Western blotting, the proteins had undoubtedly been denatured by electrophoresis on SDSPAGE. To analyze antibody reactivity with native PE and PE40, we developed an ELISA. PE or PE40 was immobilized on the ELISA plate and incubated with one of the antibodies. The antibodies that bound to these immobilized proteins were then detected by biotinylated anti-mouse immunoglobulin and avidin-biotin-peroxidase complex. All of the antibodies tested (M40-1 to M40-8) reacted with both PE and PE40 equally well when they were immobilized on the ELISA plate (data not shown). However, it was possible
Monoclonal antibody
M40-1 M40-2 M40-3 M40-4 M40-5 M40-6 M40-7 M40-8
None
Urea + DTT
pH 5.0
pH 4.0
pH
3.5
pH 3.0
94 64
AND
IMMUNITY, Jan. 1991, p. 407-414
Vol. 59, No. 1
0019-9567/91/010407-08$02.00/0 Copyright C 1991, American Society for Microbiology
Analysis of Pseudomonas Exotoxin Activation and Conformational Changes by Using Monoclonal Antibodies as Probes MASATO OGATA, IRA PASTAN,
AND
DAVID FITZGERALD*
Laboratory of Molecular Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892 Received 31 July 1990/Accepted 9 October 1990
Pseudomonas exotoxin (PE) is a protein toxin composed of three structural domains. In its native form, the toxin is a 66,000-M, proenzyme that must be activated to express full ADP.ribosylating activity. To study the process of activation and accompanying conformational changes, we have isolated 10 monoclonal antibodies to a 40,000-Mr fragment of the toxin (PE40) that exhibits full enzyme activity but lacks the toxin's cell-binding domain and contains amino acids 253 to 613 (comprising domains II, Ib, and E). By using mutant PE molecules in which all of domain I and portions of domains II, Ib, and III were deleted, the locations of the epitopes for each of the antibodies were determined. Eight of these monoclonal antibodies were further characterized. Of these eight, all reacted with soluble PE40 and an interleukin-2-PE40 conjugate, but only two reacted strongly with native soluble PE. However, all eight reacted with PE after it had been immobilized on nitrocellulose or after it had been activated to express full ADP-ribosylating activity. Antibodies were also assessed for their ability to neutralize the cytotoxic activity of either PE or interleukin-2-PE40. These antibodies should be useful as probes for monitoring the activation and processing of PE that occur during endocytosis and in determining the location of epitopes that are important for toxin activity.
Pseudomonas exotoxin (PE) is made and secreted by Pseudomonas aeruginosa and may contribute to the pathogenicity of certain clinical isolates by acting as a virulence factor (26). When present at concentrations of 1 to 10 ng/ml, PE is capable of killing mammalian cells, which it does by gaining entry to the cell cytoplasm and catalyzing the ADP ribosylation of elongation factor 2. Recent advances in the study of this toxin include the cloning and expression of the structural gene in Escherichia coli (9) and the analysis of its crystal structure by X-ray diffraction (1). The crystallography data revealed that PE is composed of three prominent structural domains (1). Recent work has focused on defining the role that each of the structural domains plays in the cytotoxic activity of the toxin (10, 25). PE binds and enters cells by receptor-mediated endocytosis and then translocates to the cell cytoplasm, where it inactivates elongation factor 2 and thereby shuts down protein synthesis (7, 12, 23). The amino-terminal domain, called domain Ta, binds to PE receptors on the surface of mammalian cells; then, after internalization via coated pits and endocytic vesicles, domain II mediates translocation to the cytoplasm; finally, the C-terminal domain, domain III, containing the ADP-ribosylating activity, inactivates protein synthesis. In addition to studies focused on toxin structure and function, PE-derived proteins have been used to make immunotoxins and other cytotoxic conjugates for the treatment of cancer and various other human diseases (8, 23). One immunotoxin, anti-Tac-PE, has been given to individuals with adult T-cell leukemia, and another, OVB3-PE, has been given to women with ovarian cancer (unpublished data). PE conjugates have also been developed for vaccine use (3, 6). From the latter studies and from our own experience in phase I clinical trials, it is clear that PE is an excellent immunogen. *
Here we describe the development of several murine monoclonal antibodies that were obtained after immunization with a truncated form of PE, termed PE40. PE40 lacks the toxin's N-terminal binding domain, is relatively nontoxic, and can be injected into animals without the need for "toxoiding." PE40 has at least one other notable property: unlike native PE, which is a proenzyme and requires treatment with urea and dithiothreitol (DTT) to express full enzyme activity (18), PE40 has full activity without any pretreatment (16). This suggests that the removal of domain I alters both toxin structure and function. When native PE is first added to cells, it is presumably still in its proenzyme state. Later, within cells, PE has to be processed and converted to a fully active form. This active form has been partially characterized and appears to be composed of a proteolytic fragment, 37 kDa in size, which is derived from the C-terminus of PE (21a). Of the eight monoclonal antibodies we have characterized, only two react with soluble native PE. However, all eight react with PE after it has been unfolded. Unlike the situation with PE, all the antibodies react with soluble PE40. Here we show that these antibodies can be used to monitor PE activation and to identify key functional epitopes. MATERIALS AND METHODS
Reagents. Hypoxanthine-aminopterin-thymidine was purchased from GIBCO, Grand Island, N.Y. Biotinylated horse anti-mouse immunoglobulin G (IgG; heavy and light chains), avidin DH, and biotinylated horseradish peroxidase H were purchased from Vector Lab, Inc., Burlingame, Calif. 2,2'Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) was purchased from Boehringer Mannheim Biochemical, Indianapolis, Ind., and O-phenylenediamine was purchased from Sigma, St. Louis, Mo. Protein A- or protein G-Sepharose was purchased from Pharmacia LKB, Piscataway, N.J., or Genex Co., Gaithersburg, Md., respectively. Murine antibody class- or subclass-specific antibodies were purchased
Corresponding author. 407
OGATA ET AL.
408
300 253
1
PE
200
100
n
INFECT. IMMUN.
i
400 365 405
500
600
613
Da 1 IuSt ala asn leu
1-3
pVC85 (PE40)
253
pMO235 408
1-3,asp
pVC7
0
1-2
pVC9 oul
1-4 pVC17
his met pCS7
1-3
264
TGFE
1-
400
FIG. 1. Schematic map of PE and mutants used for the epitope mapping of monoclonal antibodies. The map depicts the different regions of PE present in each molecule. The numbers, including those on the scale bar, represent amino acids, with the first amino acid of mature (processed) native PE numbered +1. TGFa, Transforming growth factor a.
from Southern Biotech, Assoc., Birmingham, Ala. PE was purchased from the Swiss Serum and Vaccine Institute, Bern, Switzerland. Plasmids, bacterial strains, and cell lines. The plasmids encoding various mutant forms of PE are shown in Fig. 1. The construction of the plasmids pVC85, pVC7, pVC9, pVC17, pCS7, and pCSll was described previously (25, 28). The construction of the plasmid encoding PE-His274'276'279, in which the arginines at positions 274, 276, and 279 were changed to histidines, was described elsewhere (14). The plasmid pMO235 encoding the entire domain II of PE was constructed as follows. pVC85 was partially digested with SfiI and then completely digested with PpuMI. A 2.9-kb DNA fragment was separated on a low-melting-point agarose gel, eluted, and ligated to an oligonucleotide duplex (5'-CGGCCAACTAACCCGGGTAAG-3' and 5'-GTCCT TACCCGGGTTAGTTGGCCGCGC-3'). In all the plasmids, the genes are linked to a phage T7 late promoter. For expression of these plasmids, E. coli BL21(XDE3), which contains a T7 polymerase gene fused to a lac operator and promoter, was used. Expression and purification of mutant forms of PE were done as described before (15). For the epitope mapping study of monoclonal antibodies by Western immunoblotting, crude cell extracts containing toxin proteins were also used. Gel electrophoresis and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (17). Gels containing between 10 and 17.5% polyacrylamide were used, depending on the size of the mutant form of PE. For immunoblotting, electrophoresed samples were transferred to nitrocellulose paper and processed with anti-PE antibodies as described previously (25). Isolation of monoclonal antibodies. Female BALB/c mice
(8 weeks old) were injected intraperitoneally (i.p.) with 20 ,ig of PE40 in 0.1 ml of complete Freund's adjuvant. One month later, 10 ,ig of PE40 in incomplete Freund's adjuvant was injected i.p. Two months later, 20 ,ug of PE40 was injected intravenously, and 3 days later, spleen cells (1.7 x 108) from an immunized mouse were fused to P3/x63-Ag 8.654 cells by using polyethylene glycol 4000. Hybridomas M40-1, -2, -4, -5, -6, -7, -8, -9, and -10 were isolated from this fusion.
Alternatively, 2 months after the second i.p. injection, PE40 was injected (5 ,ug intravenously plus 5 ,ug i.p.) for two successive days. Two days after the last injection, spleen cells from an immunized mouse were used for fusion, and hybridoma M40-3 was isolated. After fusion, cells were seeded in 96-well tissue culture plates and selected in culture medium containing hypoxanthine-aminopterin-thymidine. The reactivity of monoclonal antibodies to toxins was screened by a strip comb method described before (21). Briefly, 20 ng of PE40 was directly blotted onto a nitrocellulose filter cut into small strips. Strips were incubated with 2% gelatin in phosphate-buffered saline (PBS) at 37°C for 1 h (blocking) and then incubated with the hybridoma culture supernatant at 37°C for 30 min. After being washed with PBS containing 0.05% Tween 20 (T-PBS) three times, strips were incubated with biotinylated horse anti-mouse IgG (heavy and light chains) at 37°C for 30 min. Strips were then washed three times with T-PBS and incubated with avidin-biotinylated horseradish peroxidase complex at 37°C for 15 min. After being washed four times with PBS, strips were incubated with PBS containing, 3,3'-diaminobenzidine (0.5 mg/ ml) and H202 (1:2,500 dilution of 30% H202 solution). Cells secreting the desired antibodies were cloned by limiting dilution, with normal murine spleen cells used as feeder cells. Ten independent cell lines secreting anti-PE40 antibodies (M40-1 to M40-10) were established. The subtype of each monoclonal antibody was determined by enzymelinked immunosorbent assay (ELISA) with antibodies specific for classes or subclasses of murine immunoglobulins. Production and purification of monoclonal antibodies. Hybridoma cells (2 x 106 to 5 x 106) were injected i.p. into BALB/c mice that had been injected i.p. with 0.5 ml of pristane 10 days previously. After 7 to 11 days, ascites containing monoclonal antibodies were obtained. Monoclonal antibodies were purified from ascites or hybridoma culture supematants by using protein A (M40-4, -5, -8, and -10) or protein G (M40-1, -2, -3, -6, -7, and -9) column affinity chromatography. ELISA and ELISA competition assay. Microtitration plastic plates (Immuolon 1; Dynatech Lab. Inc., Chantilly, Va.) were coated with PE or PE40 (0.5 to 1.0 ,ug/ml in 0.1 M carbonate buffer [pH 9.6]) at 37°C for 1 h or at 4°C overnight. Plates were then blocked by 2% gelatin in PBS at 37°C for 1 h, washed three times with T-PBS, and incubated with anti-PE40 monoclonal antibodies. After being washed three times with T-PBS, monoclonal antibodies bound to the antigen-coated plates were detected with a Vectastain Kit (Vector Lab., Inc., Burlingame, Calif.). Briefly, the plates were incubated with horse anti-mouse IgG (heavy and light chains) at 37°C for 30 min, washed three times with T-PBS, incubated with avidin-biotinylated horseradish peroxidase complex at 37°C for 15 min, and washed six times with T-PBS. Either ABTS or O-phenylenediamine (0.5 to 1 mg/ml with 0.1 M sodium citrate buffer [pH 4.0]) was used as a substrate for peroxidase, and the A405 or A490 was measured, respectively. For an ELISA competition assay, 25 RI of competitors in various buffers containing 0.2% human serum albumin (HSA) was added to each well after the wells had been coated with antigen and blocked with 2% gelatin. Then, an equal volume (25 ,ll) of monoclonal antibody (0.2 to 0.4 p,g/ml) diluted in PBS containing 0.2% HSA (HSA-PBS) was added and incubated for 30 min at 37°C. Antibodies that bound to the coating antigen were detected as described above. For each well, relative absorbance was calculated as follows: relative absorbance (%) = [(A - Ab)/(AC- Ab) x
VOL. 59, 1991
100, where A is the absorbance of each well with competitor, AC is the absorbance of a well to which 25 ,ul of buffer solution alone is added instead of competitor (variation of the AC value was within 20% when different buffers were used), and Ab is the absorbance of a well to which 25 ,u of HSA-PBS was added instead of antibodies. Ab was usually less than 5% of AC when O-phenylenediamine was used and was less than 20% when ABTS was used. Treatment of PE with DTT or urea. The solution containing PE (5 mg/ml) or PE40 (1.5 mg/ml) was mixed with equal volumes of TE buffer (50 mM Tris, 1 mM EDTA [pH 8.0]) containing 80 mM DTT, 8 M urea, or 80 mM DTT plus 8 M urea. After incubation at 4°C for 1 h, samples were diluted more than 125-fold with HSA-PBS and used as competitors in the ELISA competition assay. Our preliminary experiments with ELISA showed that the DTT plus urea solution diluted more than 125-fold did not interfere the reactivity of our monoclonal antibodies (data not shown). Treatment of PE with low pH. PE or PE40 was incubated in MES buffer (5 mM MES [morpholineethanesulfonic acid], 5 mM citric acid, 150 mM NaCl, 0.9 mM CaCl2, and 0.5 mM MgCl2, pH adjusted with 1 M Tris) containing 0.2% HSA at various pH values for 30 min at 37°C. Then the samples were neutralized by an equal volume of 0.2 M sodium phosphate buffer, pH 6.0, and used as a competitor in the ELISA competition assay. The pH of the samples after neutralization was approximately 5.9, and in preliminary assays, it was shown that the epitopes on the toxin remained intact at pH 5.9. Also, the antibodies were fully reactive at pH 5.9. Neutralizing assay. PE at a concentration of 1 ng/ml or interleukin-2 (IL-2)-PE40 at 5 ng/ml was mixed with various amounts of each monoclonal antibody. The final antibody concentrations were 0.2, 1.0, 19, and 100 ,ug/ml. Toxicity for cells was then measured on Swiss 3T3 and HUT-102 cells for PE and IL-2-PE, respectively, as described previously (16).
RESULTS Establishment of murine hybridoma cell lines secreting anti-PE40 monoclonal antibodies. Because PE is highly toxic for mice (50% lethal dose [LD50] = 0.2 jig), it is not possible to inject large amounts of the native toxin as an immunogen. Inactivation of PE by treating it, for example, with glutaraldehyde may destroy important antigenic sites or alter the conformation of the toxin. Also, native PE is a proenzyme and undoubtedly has a different structure than enzymatically active forms. Therefore, we chose to use a 40,000Mr fragment of PE, PE40, as the immunogen (Fig. 1). PE40, which lacks domain Ia, the cell-binding domain of PE, is 250-fold less toxic than PE for mice (10). Also, unlike native PE, it has full ADP ribosylation activity (16). Spleen cells from BALB/c mice immunized with PE40 were used for cell fusion. Antibody-secreting hybridomas were then screened by a strip-comb blotting method (21). Reactivity of culture supernatants was tested by blotting samples onto nitrocellulose filter paper to which PE40 had been bound. After screening and cloning, 10 independent hybridoma cell lines secreting anti-PE40 monoclonal antibodies (M40-1 to M40-10) were established. The isotype of each monoclonal antibody was determined by using isotypespecific antibodies. M40-1, -2, -3, -6, -7, -9, and -10 were IgGl(K). M40-4 and -5 were IgG2a(K). M40-8 was IgG3(K). Location of reactive epitopes. To determine the specific regions of PE40 recognized by each antibody, PE40 and various PE-derived proteins were expressed in E. coli, subjected to SDS-PAGE, and then analyzed by Western
ANTIBODIES TO PSEUDOMONAS EXOTOXIN P!-asrmd,
('armmno acids preseWl)
t2
pVCB5 253 613 Ud
3
_
4
5
6
7
8
9
10
409
kDa -43
_ -
.'-' P4L-
---
-
_ so
_
_
S
1-2. .......
pVC9 309-613, -25.7 DVC17 381- 673'
-
--
pVC7 408 613
oM0235 `253- 364m D o? 7. a iI
_ -
*-
.
a
IV -25.7
_S
-25.7 14.3
FIG. 2. Reactivity of monoclonal antibodies to various mutants of PE on immunoblotting assay. The gene products of pVC85 (PE40), pMO235, pVC7, pVC9, and pVC17 were run on SDS-PAGE and blotted on the nitrocellulose filter, and the reactivity of each monoclonal antibody at 2 ,ug/ml was tested. Lane 1, M40-1; lane 2, M40-2; lane 3, M40-3; lane 4, M40-4; lane 5, M40-5; lane 6, M40-6; lane 7, M40-7; lane 8, M40-8; lane 9, murine IgG (2 jig/ml); lane 10, mouse anti-PE40 serum (500-fold dilution). *, Not done. The arrow shows degraded products of PE40.
blotting (Fig. 2). For this analysis, we selected 8 of the 10 monoclonal antibodies (M40-1 to M40-8) that reacted strongly in the strip-comb assay. The results are summarized in Fig. 2 and 3. All of the antibodies tested reacted strongly with both PE (data not shown) and PE40 on Western blotting (Fig. 2). M40-1, -2, and -3 reacted with domain TI of PE (amino acids [a.a.] 253 to 364), while M40-4, -5, -6, -7, and -8 did not. M40-1, -2, and -3 also reacted with a protein composed of a.a. 264 to 613 (data not shown), indicating that these antibodies reacted with an epitope(s) on the C-terminal side of a.a. 264. M40-1, -2, and -3 did not react with a protein composed of a.a. 309 to 613 that lacked the N-terminal half of domain TI, suggesting that these antibodies reacted with epitopes between residues 264 and 308 of domain TI. However, from these results, it is difficult to determine the exact location of the C-terminal boundary, since it is possible that the epitope(s) extends over a.a. 308 and the deletion of a.a. 308 or a few amino acids on the N-terminal side of a.a. 308 could destroy the epitope(s). In summary, the epitopes reactive with antibodies M40-1, -2, and -3 were mapped between residue 264 and residue 308 (Fig. 3). In addition, we tested a mutant of PE in which the arginines at positions 274, 276, and 279 were replaced by histidines (PE-His274 276'279; data not shown). M40-1 and -2 reacted strongly with this mutant protein while M40-3 did not, suggesting that at least one of these amino acids contributes to the epitope of M40-3. M40-8 reacted strongly with a protein composed of almost all of domain III (a.a. 408 to 613) (Fig. 2). The reactivity of M40-8, which was mapped between residues 408 and 613 (Fig. 3), was not analyzed further. M40-4, -5, -6, and -7 did not react with either domain TI (a.a. 253 to 364) or a protein composed of almost all of domain III (a.a. 408 to 613) (Fig. 2) or a protein made up of a.a. 400 to 613 (data not shown). These results suggested that the epitopes that reacted with these four antibodies were located between residues 365 and 399. M40-6 and -7 reacted strongly with a protein composed of a.a. 381 to 613 that lacked domain II and the N-terminal half of Ib, whereas M40-4 and -5 showed very low or no reactivity with that protein. From these results, the epitopes
OGATA ET AL.
410 9
100
INFECT. IMMUN.
200
300
500
365 405
253
1
400
600
TABLE 1. Relative affinity of monoclonal antibodies for PE after various treatmentsa
613
Relative affinity for PE (%)
PE 264 308
-0O
M40-1 M40-2 M40-3
365 399
0-0 M40-4 M40-5 381 399
M4046 M40-7
613
408 M40-8
FIG. 3. Summary of epitope mapping of monoclonal antibodies. The numbers show the amino acid number on the boundary of each epitope. The boundary was determined by either binding ( 0) or nonbinding (0) of monoclonal antibodies to mutant PE proteins, as described in the text.
of M40-6 and -7 were mapped to the C-terminal half of domain lb (between residues 381 and approximately 399). The epitopes of M40-4 and -5 were mapped in domain Ib (between residue 365 and residue 399). It is unclear whether the epitopes of M40-4 and -5 are on the C- or N-terminal side of a.a. 381 due to their marginal reactivity with a.a. 381 to 613. Characterization of antibody binding to PE and PE40 by ELISA. Next, we tested the ability of these antibodies to bind native PE or PE40. Although all antibodies reacted well with both PE and PE40 on Western blotting, the proteins had undoubtedly been denatured by electrophoresis on SDSPAGE. To analyze antibody reactivity with native PE and PE40, we developed an ELISA. PE or PE40 was immobilized on the ELISA plate and incubated with one of the antibodies. The antibodies that bound to these immobilized proteins were then detected by biotinylated anti-mouse immunoglobulin and avidin-biotin-peroxidase complex. All of the antibodies tested (M40-1 to M40-8) reacted with both PE and PE40 equally well when they were immobilized on the ELISA plate (data not shown). However, it was possible
Monoclonal antibody
M40-1 M40-2 M40-3 M40-4 M40-5 M40-6 M40-7 M40-8
None
Urea + DTT
pH 5.0
pH 4.0
pH
3.5
pH 3.0
94 64
