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Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of ricin, saporin, pokeweed antiviral
protein, diphtheria toxin, or Pseudomonas exotoxin A
(PE)2
(1). Subsequently, immunotoxins were produced using
methods of genetic engineering in which the Fv fragments of Abs were
fused to the toxin and the recombinant protein was made in
Escherichia coli (2). Our laboratory has
focused on the development of recombinant toxins in which the Fv
fragments of Abs are fused to a 38-kDa mutant form of PE
(3). PE is a 66-kDa protein composed of three domains: a
binding domain, a translocation domain, and an ADP-ribosylating domain.
Recombinant immunotoxins are made by deleting the cell-binding domain
of PE and replacing it with the Fv portion of an Ab (4).
During the past several years we have made a large number of
recombinant toxins using different Abs (5). Three of these
recombinant immunotoxins have now been evaluated in phase I trials in
patients with cancer. All the recombinant immunotoxins that have been
brought to clinical trials have been shown to cure human cancer
xenografts growing in nude mice and to be relatively well tolerated by
mice and monkeys (5). In a recently completed phase I
trial, eight partial responses have been observed in patients with
hematopoietic malignancies treated with anti-Tac(Fv)-PE38 (LMB-2).
However, side effects have been observed that cannot be attributed
to targeting IL-2R positive cells. These side effects limit the amount
of immunotoxin that can be given to humans. It seems likely that if we
can understand the mechanism or basis of the nonspecific toxicity, we
will be able to use genetic engineering to make new molecules with
fewer side effects and, therefore, we will be able to give larger doses
to humans, resulting in more responses. The toxic side effects of recombinant immunotoxins are of two types. One type of toxicity results from specific targeting of normal cells which display the same Ag as the cancer cell. The second type of toxicity is nonspecific and usually is characterized by damage to liver cells; this increases the serum levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvate transaminase (6), although other toxic effects may occur (1).
A phase I trial with anti-Tac(Fv)-PE38 has just been completed. In that trial, dose-limiting toxicity was most frequently due to liver damage. This type of toxicity is nonspecific because liver cells do not express IL-2 receptors. In the current study we have investigated the basis of the nonspecific toxicity of anti-Tac(Fv)-PE38 using mice in which liver toxicity is dose-limiting as in humans and monkeys. We have noticed that the Fv portion of anti-Tac(Fv)-PE38 has a very high isoelectric point (pI) of 10.2, whereas another recombinant immunotoxin, RFB4(Fv)-PE38, which is targeted at the CD22 Ag on human B cells, has a much lower pI of 7.67 and is extremely well tolerated by mice (7). This result suggests that the pI of the Fv portion of the recombinant immunotoxin might contribute to nonspecific toxicity because the high pI makes the protein positively charged at neutral pH and would favor binding to negatively charged groups on the surface of cells in the liver. In the current study, we have used site-directed mutagenesis and molecular modeling to decrease the pI of the Fv portion of anti-Tac(Fv)-PE38 from 10.21 to 6.82. We find that a mutant of anti-Tac(Fv)-PE38 with a pI of 6.82 has the same cytotoxic activity on target cells and the same antitumor activity as the parental anti-Tac(Fv)-PE38. However, the toxicity of this mutant immunotoxin in mice is decreased by more than 3-fold.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pI of each Fv was calculated using a program in the Genetics Computer Group (Madison, WI) package that is available through the web site http://molbio.info.nih.gov/molbio/gcglite/protform.htm. In the Fv portion, cysteines have no charge because they are disulfide bonded. These were converted to serine for the pI calculation. LMB-2 has a high pI (10.21), whereas the pI of RFB4 is 7.67.
Anti-Tac(Fv)-PE38 mutagenesis
Mutagenesis of anti-Tac(Fv)-PE38 was done by Kunkel’s method (8) with some modifications. CJ 236 cells were transformed with pRK79. After selection on Luria-Bertani plates containing 2% glucose, 30 µg/ml chloramphenicol, and 100 µg/ml ampicillin, the transformants were grown in 2x YT medium containing glucose and the antibiotics, as mentioned above, and 0.025% uridine at 37°C. At an OD600 of 0.36, the cells were infected with the helper phage M13 at a multiplicity of infection of 5. After incubation at 37°C/110 rpm for 1 h, the culture was maintained at 37°C/300 rpm for another 6 h. The bacterial cells then were precipitated by centrifugation and the phage from the supernatant was precipitated with polyethylene glycol. The single-stranded, uracil-containing DNA from the purified phage was extracted with phenol and chloroform and precipitated with lithium chloride and chilled ethanol at final concentrations of 0.8 M and 75%, respectively. This ssDNA codes for the sense strand of anti-Tac(Fv)-PE38. The following primers were used for anti-Tac(Fv)-PE38 mutagenesis: M1 VL Q1D, 5'-TGCTGGAGACTGGGTGAGAACGATATCAGAGCCGCCGCCACCCGAGCCGCCACCGCCCGAGCCACC-3'; M1 VL Q1D+VL3E, 5'-GATTGCTGGAGACTGGGTGAGTTCGATATCAGAGCCGCCGCCACCCGAGCCGCCACCGCCCGAG-3'; M1 VL S70D, 5'-GCTGATTGTGAGAGAGTAATCGGTCCCGGATCCACTGCCACTGAAGCG-3', M1 VL S100D, 5'-TTTGAGCTCCAGCTTGGTACCATCACCGAACGTGAGTGGGTA-3'; M1 VH Q1E, 5'-TGAGGCCCCAGGTTTTGCTAGCTCAGCCCCAGACTGCTGCAGATGGACCTCCATATGTATATCTCC-3'; M1 VH Q6E, 5'-TGAGGCCCCAGGTTTTGCTAGCTCAGCCCCAGACTCCTGCAGATGGACCTGCAT-3'; M1 VH Q1E+H3Q+Q5V+Q6E, 5'-TGAGGCCCCAGGTTTTGCTAGCTCAGCCCCAGACTCCACCAGTTGGACCTCCATATGTATATCTCC-3'; and M1 VH G42E, 5'-AATCCATTCCAGACCCTGTTCAGGTCTCTGTTTTACCCAGTGCAT-3'.
The primers were phosphorylated using polynucleotide kinase and T4 DNA
ligase buffer from Boehringer Mannheim (Indianapolis, IN). These
phosphorylated primers were used to introduce the mutations in the
uracil template of pRK79 using Bio-Rad (Richmond, CA) Muta-Gene kit.
The product at the end of the mutagenesis reaction was used to directly
transform DH5 competent cells. Minipreps were made from single
colonies and analyzed with the restriction enzymes, sites which had
been introduced by the primers for mutagenesis. Mutations in the clones
were confirmed by automated DNA sequencing.
Production of recombinant protein
The components of anti-Tac(Fv)-PE38 and 10 mutants were
expressed in Escherichia coli BL21(
DE3) and accumulated
in inclusion bodies as previously described for other recombinant
immunotoxins (7). Inclusion bodies were solubilized in
GuCl, reduced with DTE (dithioerythritol), and refolded by dilution in
a refolding buffer containing arginine to prevent aggregation, and
oxidized and reduced glutathione to facilitate redox shuffling
(7). Active monomeric protein was purified from the
refolding solution by ion exchange and size exclusion chromatography to
near homogeneity as described (7, 9). Protein
concentrations were determined by Bradford assay (Coomassie Plus;
Pierce, Rockford, IL).
Cytotoxicity assays
The specific cytotoxicity of the anti-Tac(Fv)-PE38 and its mutants was assessed by protein synthesis inhibition assays (inhibition of incorporation of tritium-labeled leucine into cellular protein) in 96-well plates as previously described (7, 9). The activity of the molecule is defined by the IC50, the toxin concentration that reduced incorporation of radioactivity by 50% compared with cells that were not treated with toxin. The specificity is obtained by comparing the activity toward Ag-positive cells vs toxicity against Ag-negative cells. Fresh malignant cells were partially purified from patients with B-cell leukemia and incubated with recombinant toxins as previously described (10).
Nonspecific toxicity assay
Groups of five female (
20 g) NIH Swiss mice were given
injections i.v. through the tail vein of 200 µl of escalating doses
of anti-Tac(Fv)-PE38 or its mutant immunotoxins diluted in
PBS-human serum albumin
(HSA)3 (0.2%).
Animals were observed 2 wk for mortality. The
LD50 is the calculated dose of immunotoxin that
kills 50% of the animals.
Histological study
NIH Swiss mice were sacrificed 24 h after injection of immunotoxin. Liver and kidney were fixed by 10% Formaline. Sections from each of these organs were stained with hematoxylin and eosin (H&E) and examined histologically.
Antitumor activity of anti-Tac(Fv)-PE38 and M1(Fv)-PE38 in nude mice
Antitumor activity of anti-Tac(Fv)-PE38 and M1 (Fv)-PE38 was determined in nude mice bearing ATAC4 cells. These cells (2.5 x 106) were injected s.c. into nude mice on day 0. Tumors about 50 mm3 developed in animals by day 4 after tumor implantation. Starting on day 4, animals were treated with i.v. injections of anti-Tac(Fv)-PE38 and M1 (Fv)-PE38 diluted in 0.2 ml of PBS-HSA (0.2%). Therapy was given once every other day (on days 4, 6, and 8) and each treatment group consisted of 5 animals. Tumors were measured with a caliper every 2 or 3 days and the volume of the tumor was calculated by using the formula: tumor volume (mm3) = length x (width)2 x 0.4.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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show the
LD50 in mice of three different recombinant
immunotoxins that are made with three different mouse mAbs, none of
which specifically react with mouse cells. Anti-Tac(Fv)-PE38 is
targeted at the subunit of the human IL-2 receptor (CD25) (2, 11). SS(Fv)-PE38 is targeted at an Ag termed "mesothelin"
that is found on mesothelial cells, ovarian cancers, and mesotheliomas
(9). RFB4(Fv)-PE38 is targeted to CD22. The data in Table I show that when a single dose of each of the recombinant immunotoxins
is injected i.v. in mice, the LD50s vary widely.
The most toxic is anti-Tac(Fv)-PE38 with an
LD50 of 0.34 mg/kg. The least toxic is
RFB4(Fv)-PE38 which has an LD50 of 0.81 mg/kg.
SS(Fv)-PE38 has intermediate LD50 of 0.68 mg/kg.
These three molecules are identical in the PE portion but vary in the
Fv. Thus, we concluded that these differences in toxicity were due to
some different property in the Fv. The data in Table I also show pI of
the Fv portion of these recombinant immunotoxins. They vary widely from
a pI of 10.2 for anti-Tac(Fv) to a pI of 7.67 for RFB4(Fv). The PE
portion of the molecules is rather acidic, with a pI of 5.4. Thus the
nonspecific toxicity increases with pI. The cause of death resulting
from injection of all these immunotoxins is acute hepatic necrosis.
Therefore, it seemed likely that the toxicity in mice is due to some
direct interaction of the Fv portion of the recombinant immunotoxins
with the liver. Because the liver cell membranes have a negative
charge, we hypothesized that the positive charge at neutral pH on the
Fv portion of the recombinant immunotoxins would favor higher
nonspecific binding and uptake of the recombinant immunotoxins by cells
in the liver. If this were the case, one should be able to decrease the
toxicity of anti-Tac(Fv)-PE38 by decreasing its pI by site-directed
mutagenesis of the framework regions leaving the CDRs intact and,
therefore, not affect the specific interactions of
anti-Tac(Fv)-PE38 with the human IL-2 receptor.
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These differences in positive and negative residues are responsible for
the differences in the pI between RFB4(Fv) and anti-Tac(Fv).
Therefore, we decided to make anti-Tac(Fv) more like RFB4(Fv).
Accordingly, we aligned the RFB4 and anti-Tac(Fv) sequences and
mutated several surface-exposed residues in the framework region in
anti-Tac(Fv) to make them resemble sequences in RFB4(Fv). This had
the effect of lowering the pI. Initially, we made a series of
recombinant immunotoxins in which single residues were mutated to
acidic residues one at a time. The residues in anti-Tac that were
changed are Q1, V3, S70, and S100 of VL; and Q1
and G42 of VH (Table III). In general, the residues were
changed to aspartate or glutamate. In addition, mutations H3Q, Q5V, and
Q6E were introduced in the VH to make the amino
terminus more closely resemble the amino terminus of RFB4. After these
single-residue mutants were analyzed for cytotoxicity and mouse
toxicity, two more recombinant immunotoxins (M1 and M2) were
constructed in which mutations were combined (see Table III).
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Overall, we constructed 10 mutants of anti-Tac(Fv)-PE38. The
mutations were confirmed by DNA sequencing and the recombinant proteins
were expressed in E. coli, where they all accumulated in
inclusion bodies. All immunotoxins were purified by ion-exchange and
size-exclusion chromatography from renatured inclusion bodies. Each of
the 11 immunotoxins (10 mutants and 1 wild type) eluted as a monomer
upon TSK gel-filtration chromatography and migrated as a single, major
band of about 63 kDa in SDS-PAGE (data not shown). The data in Table IV show the activity of each of the
mutant molecules on Ag-positive ATAC-4 cells that express CD25 and the
calculated pI of the Fv portion. All of the molecules constructed had
the same activity on ATAC-4 cells. Therefore, by introducing negatively
charged residues, there was no loss of specific cytotoxic activity on
target cells expressing human IL-2R. Introduction of single
negatively charged residues lead to only a small decrease in the
calculated pI. Introduction of one or two negatively charged residues
also led to a small decrease of pI from 10.2 to 9.95. Only when several
mutations were combined did the pI fall significantly. The pI fell to
6.82 for mutant M1 and to 7.76 for mutant M2.
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). Groups of five mice or more were injected with
varying doses of immunotoxin and observed for 2 wk. Almost all of the
deaths occurred within 72 h after treatment. In general, single
amino acid changes did not produce large changes in mouse toxicity,
although both the decreases and increases in nonspecific mouse toxicity
were observed by single mutations. However, when all of the mutations
were combined into one molecule to produce scM1, which had a pI of
6.82, the single-dose toxicity in mice was greatly diminished; we found
that the LD50 rose from 0.34 mg/kg to 1.22 mg/kg.
Mutant M2 has a pI of 7.76 and contains all the mutations found within
M1 except for the G42E mutation in VH. The animal
toxicity (LD50) of M2(Fv)-PE38 is 0.56 mg/kg,
which is intermediate between the toxicity of anti-Tac(Fv)-PE38 and
M1(Fv)-PE38. Liver toxicity
To determine the cause of death, mice were sacrificed 24 h
after administration of 9 µg (0.45 mg/kg) of anti-Tac(Fv)-PE38 or
M1(Fv)-PE38 and sections of the livers were prepared and stained with
H&E. In the mice treated with anti-Tac(Fv)-PE38, there was evidence
of severe hemorrhagic liver necrosis, whereas the livers from the mice
treated with M1(Fv)-PE38 appeared normal (Fig. 1). The finding of hemorrhagic necrosis
raises the possibility that the liver endothelial cells are being
damaged as part of the process of liver toxicity.
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We selected M1(Fv)-PE38 for further study because of its low
animal toxicity. Before doing further animal studies, we investigated
its cytotoxic activity on several other cell lines (Table V). HUT102 is a cell line from a patient
with adult T cell leukemia. Anti-Tac(Fv)-PE38 and M1(Fv)-PE38 had
similar cytotoxic activities on this cell line with an
IC50 value of 0.1 ng/ml. Raji is a cell line of B
cell lineage and does not express IL-2 receptors. Neither immunotoxin
was active at 1000 ng/ml. We also examined A431 cells which do not have
IL-2 receptors and are the parent of ATAC4. The
IC50 was 390 ng/ml for anti-Tac(Fv)-PE38 and
510 ng/ml for M1(Fv)-PE38. These very high IC50
values compared with the IC50 of 0.07 ng/ml on
ATAC4 cells indicate that the cytotoxic activities of both molecules
are highly specific. To determine whether M1(Fv)-PE38 would retain
cytotoxicity toward malignant target cells directly obtained from
patients, it was incubated with peripheral blood mononuclear cells from
two patients with CD25+ B-cell leukemias. In both
patients the malignant cells composed >95% of the cell sample tested.
As shown in Table V, there was no significant difference in the
cytotoxic activity of the two immunotoxins, indicating that the
mutations which lowered the pI did not impair cytotoxicity or stability
of the protein during its interaction with fresh malignant patient
cells.
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To evaluate antitumor activity, mice were implanted s.c. on day 0
with ATAC4 tumor cells and i.v. therapy was initiated on day 4 using
increasing doses of anti-Tac(Fv)-PE38 or M1(Fv)-PE38. Each agent
was given every other day for three doses. Typical tumor regression
results are illustrated in Fig. 2 for
mice treated with increasing doses of M1(Fv)-PE38. The data in Table VI shows the toxicity at each dose level
and a summary of tumor responses. The data confirm that
anti-Tac(Fv)-PE38 is much more toxic to mice than M1(Fv)-PE38. With
anti-Tac(Fv)-PE38, 1 of 15 animals died at 0.075 mg/kg x 3; 2
of 15 died at 0.15 mg/kg x 3; and all 15 died at 0.3 mg/kg
x 3. In contrast, there were no deaths with M1(Fv)-PE38 even at 0.5
mg/kg x 3. Thus, the three-dose toxicity results in nude mice
confirm the one-dose study in normal mice showing that M1(Fv)-PE38 is
much less toxic than anti-Tac(Fv)-PE38. The effect of the two
immunotoxins on tumor size is also shown in Table VI. The molecules
were equally active at the 0.075 mg/kg x 3 and 0.15 mg/kg x
3 doses, producing 6 of 15 or 7 of 15 complete regression (CR) at the
lower dose, and 11 of 15 and 14 of 15 CRs at the higher dose. At the
dose levels where anti-Tac(Fv)-PE38 caused deaths, M1(Fv)-PE38
produced 14 of 15 CRs (0.3 mg/kg) and 5 of 5 CRs (0.5 mg/kg). These
data clearly show the usefulness of being able to give higher doses of
immunotoxin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our strategy was to change framework residues so that Ag binding would
not be affected. We selected residues that differed between the Fv of
mAb RFB4 and that of anti-Tac because RFB4(Fv) has a pI of 7.67 was
much less toxic to mice than anti-Tac(Fv)-PE38 (Table II). In this
approach we only change one marginally basic residue
VH H3; this change was not expected to alter the
pI because histidine should be largely unprotonated at pH 7.2, which is
the pH at the surface of the cells in the liver. The data in Table II
show that most of the changes were from neutral to acidic residues. We
plan to investigate the effect of changing basic residues to acidic or
neutral residues in the next series of experiments. We had previously
made a compilation of the frequency with which each amino acid is
present in the framework regions of VH and
VL (13). Many residues are highly
conserved and were not changed because such mutations might affect Fv
folding or stability. We have chosen only framework residues which are
variable and which lie on the surface of the molecule so they would not
affect protein folding or stability. To examine the effects of the
change in pI on the electrostatic potential of the surface of
anti-Tac(Fv), we employed a model of the Fv portion of the Ab. Fig. 3 shows the electrostatic potential
mapped to the molecular surface of wild-type and M1-mutant models of
anti-Tac(Fv), with red designating negative and blue designating
positive. Note the large increase in the negative potential on both the
front and back sides of the M1 mutant. The figure also shows regions of
remaining positive charge, which are candidates for further
mutagenesis.
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The toxic side effects of immunotoxins in animals and humans are of two types. One side effect arises from the targeted killing of normal cells that have the same Ag as the tumor cells. The best solution to overcome this toxicity is to find a different target Ag that is not expressed on normal cells. The second type of toxicity arises from undefined nonspecific binding to normal cells. The liver is particularly vulnerable because it is susceptible to apoptosis induced by toxic substances, it has a high blood content, and its capillaries are fenestrated allowing immediate access of the high concentrations of immunotoxins that are in the blood just after injection. However, capillary damage could also occur leading to hemorrhagic liver necrosis and also contributing to the vascular leak syndrome that has been observed with immunotoxins containing ricin as well as those containing PE (17, 18). We have begun to decrease the pI of the Fvs of other recombinant immunotoxins to determine whether the decrease in animal toxicity observed with anti-Tac(Fv)-PE38 occurs with other molecules. Preliminary evidence indicates that this is the case.
In summary, we have been able to decrease the nonspecific toxicity of anti-Tac(Fv)-PE38 (LMB-2) in mice by about 3-fold without decreasing its specific cellular or antitumor activity. If this change also reduces toxicity in humans by 3-fold, we should be able to greatly increase the response to anti-Tac(Fv)-PE38 which already has shown good antitumor activity in patients with CD25+ leukemias and lymphomas (1).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: PE, Pseudomonas exotoxin A; pI, isoelectric point; HSA, human serum albumin; H&E, hematoxylin and eosin; CR, complete regression.
Received for publication June 9, 1999. Accepted for publication September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain and ricin -chain immunotoxins rapidly damage human endothelial cells: implications for vascular leak syndrome. Exp. Cell Res. 206:227.[Medline]
-Chain structure from a crystallized murine Fab': role of joining segment in hapten binding. Mol. Immunol. 18:705.[Medline]
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), 0.075 mg/kg
(
), or 0.3 mg/kg (
) of M1(Fv)-PE38 in Dulbecco’s modified PBS
containing 0.2% HSA. Control groups received carrier alone (
). No
death or toxicity was observed at these doses.