Bioconjugate Chem. 1994, 5, 40-46
40
An Immunotoxin with Increased Activity and Homogeneity Produced by Reducing the Number of Lysine Residues in Recombinant Pseudomonas Exotoxint Waldemar Debinskit and Ira Pastan’ Laboratory of Molecular Biology, Division of Cancer Biology and Diagnosis Centers, National Cancer Institute, National Institutes of Health, 37/4E16, 9000 Rockville Pike, Bethesda, Maryland 20892. Received June 7, 1993”
Pseudomonas exotoxin A (PE) is a protein composed of 613 amino acids arranged into three major, and one minor, domains. Immunotoxins (ITS) containing PE38, a mutant form of PE which lacks the cell binding domain (Ia, amino acids 1-252) and 16 amino acids from domain Ib (amino acids 365-380), are extremely potent cytotoxic agents which can cause a complete regression of various human carcinomas grown in nude mice. However, these ITS are a mixture of several different chemical forms since the coupling between the antibody and the toxin may occur between either the light or heavy chain of the antibody and one of the four primary amino groups present on the truncated toxin. To modify the toxin with heterobifunctional crosslinking reagents only at specific sites, we replaced lysines 590 and 606 with glutamines and lysine 613 with arginine (PE38QQR). We also added two different peptide sequences, each containing a lysine residue, at the N-terminus of PE38. In one of these the sequence is ANLAEEAFK (“Lys” peptide), and in the other, the sequence is LQGTKLMAEE (“NLys” peptide). The mutant toxins were coupled using a thioether linkage to monoclonal antibody B3 which recognizes an antigen present in large amounts on many human cancers. PE38QQR-containing recombinant toxins can only be linked to an antibody through the N-terminal methionine or the lysine within the peptide. B3LysPE38QQR and B3-NLysPE38QQR were four times more cytotoxic to target cells than the corresponding B3-LysPE38 and B3-NLysPE38 ITS. Furthermore, the antitumor effect of B3NLysPE38QQR was significantly greater than that of B3-NLysPE38. We conclude that B3LysPE38QQR and B3-NLysPE38QQR are more active because they are more homogenous components with all the antibody coupled to the N-terminus of the toxin and not some to the C-terminus, producing ITS with very low cytotoxic activity.
INTRODUCTION
Targeted toxins have been shown to be effective antitumor agents in animal models of solid human cancer (1). The usefulness of targeted toxins in clinical practice is in the initial phase of evaluation (reviewed in ref 1).As a prerequisite for clinical studies in humans, it is important that show to produce highly specific immunotoxins (ITS) antitumor activities in animal models. Furthermore, doselimiting side effects of ITSadministration are often due to the nonspecific toxicity of the toxin component (1). One way to minimize the nonspecific toxicity is t o decrease the amount of IT required to produce an antitumor effect. I t is also important that ITScan be produced in high yields to supply the necessary amount of drug needed for treatment at a reasonable cost. Various toxins have been utilized to construct ITS (2). These toxins are either the purified natural products from plants and bacteria or they are made as recombinant proteins and produced in E. coli. Our laboratory uses Pseudomonas exotoxin A (PE) for chemical coupling to monoclonal antibodies (MAbs) or to make recombinant + Part of this work has been presented a t the 3rd International Symposium on Immunotoxins, Orlando, FL, June 19-21, 1992. * To whom correspondence should be addressed. Phone: (301) 496-4797; Facsimile: (301) 402-1344. f W. Debinski received a postdoctoral fellowship from the Medical Research Council of Canada. Present address: Laboratory of Molecular Targeting, Research Center, Hotel-Dieu Hospital of Montreal, 3850 St. Urbain Street, Pavillon Marie de la Ferre, Montreal, Quebec, Canada H2W-lT8. e Abstract published in Aduance A C S Abstracts, November 15, 1993.
Not subject to U S . Copyright.
immunotoxins with single-chain antibodies (3). P E has a complex three-domain structure which reflects the multistep pathway by which PE kills eukaryotic cells (Figure 1). Domain Ia contains the receptor binding sequence (5-7), domain I1 is the site of a proteolytic cleavage and it is necessary for toxin translocation through an intracellular membrane into the cytosol (8,9), and domain I11is the enzymatic domain which ADP-ribosylates elongation factor-2 (EF-2)leading to the irreversible arrest of protein synthesis and cell death (6,101. To kill a cell, PE must be cleaved by an intracellular protease between arginine 279 and glycine 280 to produce a 37-kDa C-terminal fragment (8). This 37-kDa protein, composed of all of domain I11 and a portion of domain 11, is capable of penetrating into the cytosol (Figure 1). The amino acids at the C-terminus of PE, REDLK, are absolutely necessary for the cytotoxic activity, and this sequence resembles the endoplasmic reticulum retention signal, KDEL (11). Chimeric toxins and ITS containing KDEL were constructed and found to be more cytotoxic than molecules ending in REDLK (12; unpublished data). The REDLK and KDEL sequences are not necessary for the ADPribosylating activity of PE. To make conventional immunotoxins, we now use recombinant truncated forms of PE, such as PE40, which has domain Ia deleted, or PE38 which has domain Ia and 16 amino acids from domain Ib (365 to 380 of PE) deleted (13,14). The ITScontaining truncated PE are made using heterobifunctional cross-linking reagents which attach to amino groups on the N-terminal methionine of the toxin or on lysine residues. PE40 and PE38 have three lysine residues which are located in domain I11 at positions 590,
Published 1994 by American Chemical Society
Bioconjugate Chem., Vol. 5, No. 1, 1994
Immunotoxin with Increased Activity and Homogeneity
41
MATERIALS AND METHODS PE
M
PE38
0 . NLysPE38
NLysPE38-
MLOGTKLMAEE
0 . MLOGTKLMAEE
QQR
0.. ~
5
a Q
~
~
*
~
590, 6 0 6 ~ 6 1 3
Residue that can react with cross-linkei
--
Processing site in PE
Figure I. Schematic drawing of Pseudomonas exotoxin A (PE) and ita truncated forms. Circles correspond to the structural domains of PE: domain Ia is a binding domain (aa1-252),domain I1 contains the site of proteolytic cleavage (aa 253-4041, and domain I11 is an ADP-ribosylating enzyme (aa 405-613). The dashed line indicates the cleavage site after arginine 279 in PE. N-terminal sequences, given in a single letter code, correspond to the amino acids added to the PE38 sequences. Also listed are the positions of the three lysine residues in domain I11at positions 590, 606,and 613;amino acid 613 is the last residue in PE.
606, and 613 ( 4 ) . An additional lysine residue has been introduced within a nine amino acid peptide (“Lys“ peptide) added to PE40 at its N-terminus to form LysPE40 (13). LysPE40 is conjugated to antibodies more easily than PE40, and the resulting ITS are more active (13). Another version of the toxin has been recently made which has one lysine residue in a 10 amino acid peptide preceding PE40 (“NLys” peptide); it has been termed NLysPE40 (15). To avoid chemical modification of domain I11with crosslinking reagents, which should produce inactive or less active ITS by interfering with the ability of the 37-kDa fragment to be translocated to the cytosol, we changed the lysines at positions 590, 606, and 613 to other amino acids using site-directed mutagenesis. In studies with an antibody directed against the human transferrin receptor (HB21),PE40 with mutations in the lysines in the carboxyl terminal portion of the toxin was conjugated through a disulfide bond to HB21 and produced a more cytotoxic IT and at higher yields (16). In addition, we noticed that some forms of the mutant PE40 molecules were produced in better yields in E. coli than others with lysine residues untouched. In the present work, we tested the following: (i) if the action of the ITS containing selectively modified forms of PE38 had a better antitumor activity in mice, (ii) whether the peptides added at the amino terminus of PE40 and NLysPE40 (or PE38) and elimination of lysine residues in truncated toxins are responsible for their better expression in E.coli, and (iii) whether it is possible to use a thioether rather than a disulfide bond to combine the toxin with the antibody and to retain the higher cytotoxicity of conjugates.
~
Bacterial Strains and Plasmids. Plasmids encoding various forms of recombinant truncated PE are indicated in Table 1. Plasmid pJBlPE38 encodes LysPE38, and it has been previously described (13). Plasmid pMS8-38 encodes NLysPE38; it was prepared by subcloning a 1013 bp fragment cut with SalI, EcoRI restriction enzymes from plasmid pJBlPE38 into a plasmid pWD402 (16) cut with the same enzymes. Plasmid pWD402-38, encoding LysPE38QQR, was formed by ligating a 460 bp DNA excised with BamHI and EcoRI from plasmid pWD402 to plasmid pJBlPE38 digested accordingly. Finally, plasmid pMS8-38-402, encoding NLysPE38QQR, was produced as plasmid pWD402-38 with a difference that the vector ~ 3 ~ ’ ~ to which the BamHI, EcoRI 460 bp fragment was ligated derived from the plasmid pMS8-38. The cloning procedures for other plasmids encoding proteins listed in this work are available from the authors on request. The plasmids were propagated in the HBlOl strain of E.coli and expressed in strain BL21 (XDE3) as described (18). Expression and Purification of Recombinant PE38s. Bacteria of the BL21 (XDE3) which carry a T7 DNApolymerase gene in a lysogenic and inducible form were transformed with plasmids encoding various forms of truncated PE. Transformed BL21 were grown in LB broth in small-scale cultures (5mL) or in super-broth in cultures from 1to 10 L. Cells were allowed to grow to absorbances of 0.5 (5-mL cultures) to 10.0 (fermentor runs) at 650 nm when l-thio-@-D-galactopyranosidewas added to a final concentration of 1mM. Cells were harvested 90-120 min later and centrifuged to give a bacterial pellet. Then, the bacteria were osmotically shocked in order to prepare the periplasm in which all forms of PE38 were predominantly found. PE38s were purified using a Pharmacia fast protein liquid chromatography (FPLC) system as previously described (15). The toxins were purified to homogeneity and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were detected by staining with Coomassie-Blue or by immunoblot analysis which was performed using polyclonal antisera to PE. Construction of Immunoconjugates. To make ITS, we used essentially the same protocol as previously described (15). Briefly, monoclonal antibody B3 was modified with a 6-fold excess of 2-iminothiolane (2-IT) to provide sulfhydryl groups (19). The toxins were derived with a 10-fold excess of succinimidyl 4-W-maleimidomethy1)cyclohexane-l-carboxylate(SMCC) to provide maleimides ready to react with thiols present on the derivatized antibody (20). The ITS were purified by twostep liquid chromatography (13). Protein Synthesis Inhibition Assay. ITSwere tested on the epidermoid carcinoma A431, breast carcinoma MCF-7, and several other carcinoma cell lines. Their activities were determined by measuring inhibition of [3H]leucine incorporation into cells (6). ID50 indicates the concentration of IT at which the isotope incorporation fell by 50% when compared to nontreated cells. ADP Ribosylation Assay. ADP ribosylation activity was assayed by the method of Collier and Kandel (21). Briefly, wheat germ extract was used as a source of EF-2. Samples of recombinant toxins were diluted 1:20 in PBS/ 0.02 % BSA directly before the assay. [I4C]NAD provided labeled ADP ribose for transfer to EF-2. After 15 min of incubation, the samples were treated with trichloroacetic acid, and the precipitates were counted in a liquid scintillation counter.
Deblnski and Pastan
Bb~0n/u@¶t8Chem., Vol. 5, No. 1, 1994
42
T
P
T
P
T
P
T
P
T
P
T
P
T
P
kDa 7244
28
+
I
COOMASSIE-BLUE STAINING
A
kDa 72-
28
I
WESTERN BLOT
I
Figure2. SDS-PAGE and immunoblot analysis of the truncated recombinant forms of P E performed under nonreducing conditions: T, total cell pellet; P, periplasm. An equivalent amount of proteins was added on each lane: 2.4 hg for T and 1.4 pg for P.
Treatment of Nude Mice Bearing Human Epidermoid Carcinoma Tumors. A431 cells were injected subcutaneously on day 0 into female nude mice (four to five mice per group). Tumors developed in all injected animals, and the tumor size reached about 5 X 5 mm on day 4. The mice started to receive ITSintraperitoneally (ip) on day 4, and the treatment continued on days 6-9 or the treatment started on day 5 and continued on days 6-8. Tumors were measured with a caliper, and the formula for tumor volume calculation was as previously reported (13). LDm were established in FVB/NR and Balb/c female mice that received a single ip injection of ITS and were observed for 3 days afterwards. RESULTS
Expression of Various Truncated Forms of PE in E. coli. We attempted to engineer recombinant forms of PE that would be expressed at high levels, exported predominantly into the periplasm, and suitable for modification with heterobifunctional cross-linking reagents at specific sites in the process of conjugation with antibodies. The first form of PE40 we investigated, LysPE40, has a nine amino acid extension containing an additional lysine added at its amino end to facilitate its chemical modification (13). It was expressed in E. coli together with a protein of molecular weight around 30 kDa which was not recognized by polyclonal antisera against PE and was probably p-lactamase (Figure 2). The same toxin but with amino acids 365-380 deleted, LysPE38 was expressed at lower levels than LysPE40. In both cases most of the toxin was exported into the periplasm. Interestingly, when the three C-terminal lysines were changed so that there were glutamines at positions 590 and 606 and arginine at position 613, the expression of these toxins and the amount in the periplasm visibly improved (Figure 2). The QQR substitutions did changethe mobility of the toxins on SDSPAGE gel. Very little LysPE40QQR or LysPE38QQR
appears in the medium (not shown). NLysPE40 and NLysPE38 which contain a different sequenceat the amino terminus than LysPE4O or LysPE38 (Figure 1) are expresseda t high levels, and most of the protein is exported into the periplasm. Thus, the addition of the N-terminal peptide preceding domain I1 and I11 in NLys-toxin (MLQGTKLMAEE; Figure 1) preserves its export into the periplasm. Growth of E. coli with PE38 Toxins and a Yield of Toxin. The differences in behavior in E. coli producing various PE38 toxins is summarized in Table 1. BL21 cells transformed with plasmids encoding LysPE38 and LysPE38QQR have doubling times of around 1h. On the contrary, cells containing plasmids encoding NLysPE38 and NLysPE38QQR have a doubling time of 34 and 30 min, respectively. More importantly, we obtained much more purified NlysPE38QQR than LysPE38 from a typical fermentor run performed under identical conditions (Table 1). Preparationof B3-PE38s ITS. It has been previously found that ITSmade with PE38 are more active than those made with PE40 (unpublished data). Therefore, B3 was coupled to LysPE38, LysPE38QQR, NLysPE38, and NLysPE38QQR as described in the Materials and Methods. All possible care was taken to maintain identical coupling and purification conditions to make all four ITS. The Mono-Q and TSK chromatography profiles of all four ITSwere indistinguishable (data not shown). However, the final yield of IT tended to be higher for NLysPE38and NLysPE38QQR-containingconjugates than that for the Lys-toxin counterparts (33 and 25% vs 20 and 18% of starting materials, respectively; Table 1). More experiments are needed to establish statistically meaningful differences between the yields of various ITS. It is noteworthy that we could further improve the yield of IT up to 60% of the original material using NLysPE38QQR. The SDS-PAGE profiles of purified ITSwere very similar for all the B3 ITS under both nonreducing and reducing conditions (data not shown). Cytotoxic Activities of B3 ITS. We compared the cytotoxic activities of B3 conjugates on A431 epidermoid carcinoma cells. As shown in Figure 3, B3-LysPE38 and B3-NLysPE38 were very active with an IDm of 4 ng/mL (21 pM). However, the two conjugates containing PE38QQR were even more active 0, < 0.02; n = 5-7; Student's t-test); the ID@ averaged 1 ng/mL (5.2 pM). These results show that eliminating lysine residues at the C-terminus of the toxin increases significantly the cytotoxicity of resulting ITS. Furthermore, this increased activity is not due to the precise sequence of amino acids at the amino terminus of PE38. Toxicity of B3-PE38s in Mice. We determined the L D m for the B3 ITS in mice (Table 1). We found that L D m for B3-LysPE38 and B3-NLysPE38 were 112.5pg/ mouse and were not distinguishable from each other when given as a single ip injection. B3-NlysPE38QQR was more toxic to mice with an LDw of 87.5 pg/mouse (30% increase). Antitumor Activities of B3-PE38 Immunotoxins. Sinceour goal was to prepare more active antitumor agents, we tested B3 coupled to two forms of NLysPE38 on A431 solid tumor xenografts in nude mice. The mice were injected with A431 cells on day 0 and received treatment of five injections of 5, 7.5, and 10 pg per day of either B3-NLysPE38 or B3-NLysPE38QQR, starting on day 4 when tumors reached 40-50 mm3 (Figure 4A). B3NLysPE38caused a significantantitumor effect a t alldoses used, but it did not produce a complete regression of the tumors at up to 10pg per day (5Opgtotal dose). In contrast,
Immunotoxin with Increased Activity and Homogeneity
BioconJugate Chem., Vol. 5, No. 1, 1994 43
Table 1. Various Forms of PE38 and Their Chemical Conjuaates with MAb B3 ~~
~
toxin (plasmid) LysPE38 (~JBlpE38) LysPE38QQR (pWD402-38) NLysPE38 (pMS8-38) NLysPE38QQR (pMS8-38-402)
doubling time of E. coli (min) 60
efficiency of expression, (mg protein/OD x vol)b 2.6
~~~
final yield, (% starting material) 20
B3 immunotoxins IDw on A431 cells (ng/mL) 4
L D d (valmouse) 112.5
60
18
1
34
33
4
112.5
25
1
87.5
30
14.8
FVB/NCR female mice; average from two experiments. OD: optical density. Vol: volume of culture. 120
0 B3-LysPE38
A
I
100
v,
n
04 .1
. -...... I
1
. .......
I
. ......9
10
100
"E E u
W
Concentration (ng/ml)
Figure 3. Inhibition of protein synthesis in A431 cells by B3 ITS purified on a TSK size-exclusion column. The interrupted line shows 50% of [SHlleucineincorporation. Isotope incorporation was measured as described in the Materials and Methods. The points correspond to the average of determinations performed in triplicates.
5
40 20
0 0
8
K
10
12
14
16
0
E
B
3
I-
\
500
B3-NLysPE38QQR eliminated the tumors at a dose as low as 5 pg per day for 5 days (25 pg total dose). Injections of 7.5 and 10 pg per day also completely eliminated the tumors (not shown). This experiment demonstrated that B3-NLysPE38QQR had a greater antitumor potency than B3-NLysPE38. Since the conjugates with NLysPE38QQR exhibited higher toxicity in mice than those containing NLysPE38, we performed another experiment on A431 xenografts in which the doses of the ITS were established on the basis of their LDm values. The mice bearing the A431 tumors were injected with doses corresponding to 5,10, and 15% of their LDM (Figure 4B). When B3-NLysPE38QQR was injected for 4 days at 5% of the LDSOdose (4.4 pgI mouse/day; total dose 17.6 pg) complete tumor regression occurred in all animals. As expected, the higher doses of this IT produced the same effect, B3-NLysPE38 was a less potent antitumor agent. Even at a dose of 15% of the LDm complete tumor regression occurred in only 80 % of the treated mice (16.8 pglmouselday; 67.2 pg total dose). Toxicities of the Toxins Containing "QQR"Mutations. To rule out the possibility that the amino acid changes at the C-terminus of the PE38 toxins increased toxin activity directly instead of preventing coupling to the carboxylterminal we performed several control studies. We tested the cytotoxicity of PElPE38 and their QQR derivatives on different cancer cell lines (Figure 5). P E and PEQQR had the same toxic activity on MCF-7 cells (Figure 5A). Similar results were obtained on CRL-1739 gastric carcinoma and LNCaP prostate carcinoma cell lines (data not shown). Furthermore, LysPE38QQR and
A AAAA
2
v)
-
B3-NLySPE38
5 10 15
9 u u4
200
4
T
100
0 0
2
4
6
8 10 12 14 16 18 20 22 24 26
AAM
DAYS Figure 4. Antitumor effect of B3-NLysPE38 (open symbols) and B3-NLysPE38QQR (full symbols) on A431 xenografts in nude mice. A431 cells were inoculated subcutaneously on day 0 (3 X 1oBcells/mouse), and the mice were treated ip on the days indicated by the arrowheads. In A, equal amounts of B3NLysPE38 and B3-NLysPE38 QQR were injected into animals, and the numbers (5,7.5,10) correspond to pg/mouse/day. SEs are shown as vertical bars for a l l data points with the exception of animals receiving 7.5 pglday of B3 IT. In B, the mice received equivalent doses (5,10,15%/mouse/day) according to the LDm values of both ITS. SEs are shown for the control animals and for 5% LDd/mouse/day of both B3-NlysPE38 and B3NlysPE38QQR treated mice.
NLysPE38QQR did not appear to show any greater nonspecific toxicity than that exhibited by LysPE38 and
44
Debinski and Pastan
Bioconjugate Chem., Vol. 5, No. 1, 1994 8000 '
6000
.
0 0
NLysPE38 NLysPE38QQR
4000
20
2000 I
*
PEQQR
.1
1
0
10
100
1000
.01
.1
1
10
100
ng/tube Figure 6. ADP ribosylating activities of NLysPE38 and NLysPE38QQR. Eachresultis the mean of three determinations.
ADP Ribosylating Activities of Toxins and ITS. Since the changes at the C-terminus of PE38 might influence the ADP ribosylating activity of domain I11 we measured the ADP ribosylating activity of NLysPE38 and NLysPE38QQR. The toxins were used at various amounts per reaction. We did not observe any appreciable difference between the enzymatic activity of NLysPE38 and NLysPE338QQR (Figure 6). Since the coupling of NLysPE38QQR to MAb B3 may result in a different ADP ribosylating activity than that of NLysPE38 conjugated to B3, both B3 conjugates were tested for their ADP ribosylating activities and no differences were observed. 120
I
100 80 60 40
20
0 .1
1
10
100
1000
10000
Concentration (ng/ml) Figure 5. Cytotoxicity and nonspecific toxicity in MCF-7 (A and B) and in A431 (C) cells of PE and various forms of PE38, respectively. The interrupted line shows 50 % [3Hlleucine
incorporation.
NLysPE38 on MCF-7 cells (Figure 5B). If anything, the QQR derivatives were slightly less cytotoxic. We evaluated the effects of the various toxins on the A431 cells which had been used in the antitumor experiment and found that P E had the same activity as PEQQR and that NLysPE38 had the same activity as NLysPE38QQR (Figure 5C). We also found that in mice PE and PEQQR had the same LD60 and that NLysPE38 and NLysPE38QQR also had the same LD50 (17.5 kg/mouse).
DISCUSSION We have made ITS composed of MAb B3 coupled to various forms of recombinant PE38 with different N- and C-terminal ends. ITScontaining forms of PE38 with lysine residues substituted with Gln at positions 590 and 606 and Arg at position 613 at the C-terminus (QQR) were more cytotoxic to cancer cell lines than ITS containing a wild-type carboxyl terminus. On the other hand, changes in the extension peptide at the amino end of the toxin did not have any impact on the activity of the ITS. Moreover, the higher cytotoxicity of B3-NLysPE38QQR was translated into a better antitumor effect in nude mice bearing human xenografts. Coupling of the N-Terminus of the Toxin to MAb Increases the Cytotoxic Activity of IT. When B3 was coupled to NLysPE38QQR through a noncleavable linker, the IT was more active than B3-NLysPE38 prepared in the same way. The same phenomenon was observed with a LysPE38 version of the toxin and found for other monoclonals, such as C242, directed against colorectal cancer and mono- and divalent versions of HB21 (15,22; unpublished observation). We have demonstrated that the QQR mutation at the C-terminal end does not increase the cytotoxicity of PE, as has been observed with changes in the last five amino acids in PE (11).The most plausible explanation for higher cytotoxicity of ITS with QQR is that Lys- or NLysPE38QQR is chemically derived at one site at the amino terminus prior to the processing site of PE which lies between amino acids 279 and 280 (Figure 1). The proteolytic cleavage inside the cells produces a 37-kDa (or 35-kDa in case of PE38) fragment which must be translocated into the cytosol. If the toxin is coupled to the light or heavy chain of the antibody by its carboxyl end, a large protein would be produced that would be
Bioconjugate Chem., Vol. 5, No. 1, 1994
Immunotoxin with Increased Activity and Homogeneity IMMUNOTOXlNS CONTAINING PE38
45
N-terminus of the toxin more similar to the classic pattern of signal sequences. The N-terminal amino acids in PE38, LysPE38, and NLysPE38, with the charged residues in bold type, are as follows: NH, MAEGGSLAALTAHQACHLPL... - PE38 NH, MANLAEEAFKGGSLAALTAH ... - LysPE38 NH, MLQGTKLMAEEGGSLAALTA ... - NLysPE38
ACTIVE INACTIVE Figure 7. Schematic drawing of the structure of ITScomposed of a MAb and either PE38 or PE38QQR MAb, Y letter; H, heavy chain; L, light chain. All other symbols are as in Figure 1. Note that the toxin can also be linked to the light chain.
difficult to translocate (Figure 7) even if larger, e.g., 62kDa, proteins have been shown to translocate to the cytosol (23). In addition, coupling to the C-terminus may prevent the REDLK sequence from bringing the toxin to the endoplasmic reticulum. The 35-kDa fragment derived from NLysPE38QQRwould be neither chemicallychanged nor attached to a chain of the antibody thus leaving the fragment free to reach the endoplasmic reticulum and translocate into the cytosol. Thus, because of the increased homogeneity, B3-NLysPE38QQR would be more active. On the contrary, B3-PE38s are a mixture of conjugates between the N- and C-terminal ends of the toxin and the antibody chains (Figure 7). "QQR"Mutations Increase the Cytotoxicity of ITS by a Mechanism Different from KDEL Mutations or Mutations that Eliminate the Need for Toxin Proteolysis. The increase in the potency of PE resulting from introducing the KDEL sequence at its C-terminus makes PE-containing ITS more active in vitro but also more toxic in animals (R. Kreitman, L. Pai, and I. Pastan, unpublished results). Even though B3-NLysPE38QQR was somewhat more toxic to animals than B3-NLysPE38, its therapeutic efficacy was improved. One reason is that B3-NLysPE38QQR does not compete for B3 binding sites with inactive molecules of IT, as in the case of B3-LysPE38. B3-LysPE38 molecules that are inactive on tumor cells still may be toxic to the liver. PE38s need to be proteolytically processed inside cells (Figures 1and 7). One may argue that by eliminating the need for the proteolysis step it would be possible to increase the supply and subsequent cytotoxicity of the cytosoltargeted fragment of the toxin. Our laboratory has recently engineered a PE35 molecule which like the Ricin A chain does not require proteolysis for activation and initiation of translocation (24). However, the cytotoxicities and antitumor activities of ITS containing PE35 or NLysPE38QQR are similar (W. Debinski and I. Pastan, unpublished results). Thus, the processing of PE38 attached to B3 is not a rate-limiting step in its cytotoxicity on several cancer cell lines tested. Export of Truncated PE Toxins into the Periplasm of E. coli May Be Related to a Negatively Charged N-Terminus. NLysPE38 and NLysPE38QQR are produced in large amounts in E. coli and easily purified to near-homogeneity. It has been previously noted that PE40 is almost completely exported into the periplasm (18). The N-terminal sequence of PE40 does not, however, resemble classic signal sequences that enable bacterial proteins to be exported out of the cytoplasm (25). Introduction of "Lys" and "NLys" peptides does make the
Thus, these PE38s have a negatively charged N-terminus, and the charge is confined to the amino end or to the middle of the first 20-25 N-terminal amino acids. Recent reports suggest that a single net negative charge present in leader sequences of venoms may play a role in their export (26). All our PE38 versions of PE also have a single net negative charge within their 25 amino-terminal amino acids, as did the first form of PE40 produced in this laboratory (18). All of them have glutamic acids present near the amino end. It has also been found that a glutamic acid neutralizing the positive charge of either arginine or lysine had a strongly beneficial effect on protein export in prokaryotes (27). This negative charge provided by the glutamic acid could represent a favorable feature for some proteins to be transported through the membranes. SUMMARY
ITS containing selectively modified PE38, such as LysPE38QQR or NLysPE38QQR, are more active agents than their counberparts with a wild type c-terminus due to an increase in the specific potency of the IT. NLysPE38 or NLysPE38QQR can be produced in large amounts so that material for clinical trials can be readily obtained. ACKNOWLEDGMENT
We thank E. Lovelace and A. Harris for assistance with tissue cell culture and A. Jackson and J. Evans for secretarial help. LITERATURE CITED (1) Pastan, I., and FitzGerald, D. (1991) Recombinant toxins for
cancer treatment. Science 254, 1173-1177. (2) Frankel, A. E., Ed. (1988) Immunotoxins, Kluwer Academic Publishers, Dordrecht, Netherlands. (3) Pastan, I., Chaudhary, V., and FitzGerald, D. (1992) Recombinant toxins as novel therapeutic agents. Ann. Rev. Biochem. 61, 331-354. (4) Gray, B. L., Smith, D.'H., Baldridge, J. S., Harkins, R. N., Vasil, M. L., Chen, E. Y., and Heyneker, H. L. (1984) Cloning, nucleotide sequence and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 81, 2645-2649. ( 5 ) Allured, V. S., Collier, R. J., Carroll, S. F., and McKay, D. B. (1986) Structure of exotoxin A of Pseudomonas aeruginosa a t 3.0-Angstrom resolution. Proc. Natl. Acad. Sci. U.S.A.83, 1320-1324. (6) Hwang, J. D., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987)Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E . coli. Cell 48, 129-136. (7) Jinno, Y., Chaudhary,V. K., Kondo, T.,Adhya, S.,FitzGerald, D. J., and Pastan, I. (1988) Mutational analysis of domain I of Pseudomonas exotoxin: Mutations in domain I of Pseudomonas exotoxin that reduce cell binding and animal toxicity. J . Biol. Chem. 263, 13203-13207.
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