(Received for publication, July 5, 1995; and in revised form, August 25, 1995)
From the
Diphtheria toxin (DT) is often used in the construction of
immunotoxins. One potential problem using DT-based immunotoxins is the
pre-existing anti-DT antibodies present in human blood due to
vaccination. The present study examined the effect of human serum with
pre-existing anti-DT antibodies on the toxicity of UCHT1-CRM9, an
immunotoxin directed against CD3 molecules on T-lymphocytes. Sera with
detectable anti-DT antibodies at 1:100 or greater dilutions inhibited
the immunotoxin toxicity. Experiments with radiolabeled UCHT1-CRM9
indicate that anti-DT antibodies partially block its binding to the
cell surface as well as inhibit the translocation from the endosome to
the cytosol. The inhibitory effect could be adsorbed using a
full-length DT mutant or B-subfragment. A C-terminal truncation mutant
could not adsorb the inhibitory effect, suggesting that the last 150
amino acids contain the epitope(s) recognized by the inhibitory
antibodies. Therefore, an anti-CD3 single-chain immunotoxin, sFv-DT390,
was made with a truncated DT. The IC of sFv-DT390 was 4.8
10
M, 1/16 the potency of the
divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicity was only
slightly affected by the anti-DT antibodies in human sera.
Mutated full-length and truncated diphtheria toxin (DT) ()molecules are used for making immunotoxins. These
immunotoxins show strong cytotoxic effects to their target cells, and
some of them have already been used in clinical
trials(1, 2, 3, 4, 5, 6, 7) .
Previously, our laboratory constructed an immunotoxin directed against
the CD3
molecule of the T-cell receptor complex, a pan T-cell
marker. This construct is made with a monoclonal antibody of
mouse-origin, UCHT1, and a binding site mutant of DT, CRM9 (8) . The immunotoxin, UCHT1-CRM9, is capable of regressing
established xenografted human T-cell (Jurkat) tumors in nude
mice(9) . A rhesus monkey analog of UCHT1-CRM9, FN18-CRM9, was
capable of not only depleting circulating T-cells but also depleting
resident T-cells in the lymph nodes. (
)This immunotoxin also
delayed skin allograft rejection as compared to antibody treatment and
non-treatment controls. FN18-CRM9 has also been used as an adjunct in
inducing tolerance to mismatched kidney transplants(24) .
In
contrast with ricin and Pseudomonas exotoxin based
immunotoxins, there is a potential problem using UCHT1-CRM9, or other
DT-based immunotoxins, in the treatment of human diseases. Most people
have been immunized against DT. Therefore these people have a
pre-existing anti-DT antibody titer which could potentially inhibit or
alter the efficacy of these immunotoxins. This limitation also occurred
in our rhesus monkey studies. FN18-CRM9 could deplete T cells in the
blood, but to a much lesser extent in animals with anti-DT antibodies,
and the T cells repopulated several days earlier compared to those
monkeys without anti-DT titers. In order to overcome this
antibody mediated inhibition, we undertook the first examination of the
effect and the mechanism of human sera containing anti-DT antibodies on
UCHT1-CRM9 toxicity. A DT point mutant, a truncation mutant, and DT
subfragments were used in an attempt to neutralize the anti-DT effect
in human sera. Based on the neutralization data, a single-chain
immunotoxin was constructed with a C-terminal deletion mutant of DT
which could potentially bypass the inhibitory effect of the
pre-existing anti-DT antibodies.
Figure 1:
The epitopes involved in human
serum's inhibition of toxicity lie in the last 150 amino acids of
DT. A schematic diagram of the DT mutants CRM9, CRM197, and MSP5
is presented (A). The A- and B-subfragments and their relative
size and position are shown. The filled circle represents a
point mutation as described in the text. Goat (B) or human (C) serum (human serum was a pool from all samples with
positive ELISA for anti-DT antibodies) was incubated with increasing
molar concentrations of CRM197 (
), MSP
5 (
), or the
B-subfragment (
) of DT for 30 min at room temperature. To this
reaction, UCHT1-CRM9 was added to a final concentration of 1
10
M. This mixture was then diluted
10-fold onto Jurkat cells in a protein synthesis inhibition assay as
described under ``Materials and Methods.'' Immunotoxin
incubated with medium only inhibited protein synthesis to 4% of
controls. The results are representative of two independent
assays.
Figure 2:
sFv-DT390 maintains specificity for the
CD3 complex but is 16-fold less toxic than UCHT1-CRM9 to Jurkat cells. A, increasing concentrations of sFv-DT390 () or
UCHT1-CRM9 (
) were tested in protein synthesis inhibition assays
as described under ``Materials and Methods.'' The results are
an average of four separate experiments. B, increasing
concentrations of UCHT1 antibody were mixed with a 1
10
M UCHT1-CRM9 (
) or 3.3
10
M sFv-DT390 (
) and then added to
cells for a protein synthesis inhibition
assay.
The current investigation is the first analysis on the effect
of pre-existing anti-DT antibodies on DT-based immunotoxins and to
determine the mechanism of the observed effect. Our results indicate
that the pre-existing anti-DT antibodies present in human serum inhibit
the toxicity of the immunotoxin UCHT1-CRM9. This inhibition of toxicity
was also observed with goat anti-DT serum, however, less goat serum was
needed to completely inhibit toxicity. The experiments were designed in
such a way to mimic the in vivo situation. The peak
concentration of circulating immunotoxin currently being tested in
animal models is 1 10
M. The
immunotoxin concentration incubated with the 1:10 dilution of human
serum was 1
10
M, thus
approximating in vivo conditions. The inhibition of toxicity
correlates with the serum antibody levels as determined by ELISA (Table 1), indicating that sera with higher anti-DT titers have a
stronger inhibitory effect. Similarly, the goat anti-DT serum which
gave the highest ELISA value could be diluted 10,000 times and still
completely inhibited UCHT1-CRM9 toxicity. Since this correlation
exists, there is no indication that any other component of the serum
inhibits the toxicity of UCHT1-CRM9. Furthermore, our data show that a
titer of 1:100 dilution is necessary for an inhibition of the
immunotoxin toxicity. This is in agreement with data from a clinical
trial(20) . A construct in which the first 486 amino acids of
DT were fused to interleukin-2, DAB
IL-2, was used in
lymphoid malignancy patients. A partial response to
DAB
IL-2 was observed in several patients who had a
anti-DT titer below 1:100 dilution prior to the treatment.
Intoxication of cells by immunotoxins can be subdivided into four general stages: 1) specific binding to the cell surface, 2) endocytosis into the cell, 3) translocation of enzymatic domain of the toxin out of the endosome, and 4) enzymatic inactivation of the target molecule. The results presented indicate that, while the amount of immunotoxin reaching the cell surface is lower in the presence of serum, the same percentage of bound immunotoxin is endocytosed. Taking into account the reduced amount of immunotoxin bound to the cell, the amount of endocytosed immunotoxin should intoxicate the cells to below 25% of controls. However, the immunotoxin had no effect on protein synthesis in the presence of serum containing anti-DT antibodies. Since the A-subfragment of DT could not adsorb the protective effect of serum while the B-subfragment could, the effect of serum is not likely to be at the level of inhibiting enzymatic activity of the toxin. Therefore, it suggests that the anti-DT antibodies affect the translocation of the A-subfragment into the cytosol.
CRM197, B-subfragment, and MSP5
could adsorb the protecting anti-DT antibodies from the goat and rhesus
monkey (data not shown) sera. However, among the 3 DT mutants,
MSP
5 could not prevent the UCHT1-CRM9 toxicity in the presence of
the human sera, showing a difference in the anti-DT antibody repertoire
among humans, goat, and rhesus monkeys. This difference does not seem
to be due to immunization routes, because monkeys used in the present
study were not immunized for DT and presumably acquire the antibodies
after a natural infection with toxigenic strains of C.
diphtheriae. Although there were reports showing that rhesus
monkeys and humans shared a similar antibody repertoire(21) ,
our results suggest that one must analyze the effect of antibodies from
the host for whom immunotoxin treatment is intended.
To overcome the
blocking effect of the pre-existing anti-DT antibodies in human sera,
there are basically two pathways existing. One is to neutralize the
antibodies with non-toxic DT mutants, ()and the other is to
modify the DT structure used for making immunotoxin(3) . The
antibody neutralization pathway has been tested in our monkey studies
of FN18-CRM9 treatment. A 100-fold higher amount of CRM197 was injected
5 min before FN18-CRM9 to adsorb the pre-existing antibodies in 2
monkeys who had an anti-DT titer at 1:1,000 dilution. In one monkey the
T cell depletion was as good as in monkeys without anti-DT titers. The
other monkey died due to multiple kidney infarcts. It is possible that
this condition resulted from immune complex disease precipitated by the
neutralization procedure. Thus, serum neutralization may be a
potentially dangerous process. Our results showed that although
antibodies against both A- and B-subfragments existed in human sera,
MSP
5 could not neutralize the pre-existing protective anti-DT
antibodies, and therefore could not prevent the inhibition of the
cytotoxicity of UCHT1-CRM9. However, it did block the inhibitory effect
of the goat and monkey sera. This prompted the construction of the
recombinant immunotoxin, sFv-DT390. The IC
of sFv-DT390 is
4.8
10
M, 1/16 as potent as
UCHT1-CRM9. Like many other single-chain constructs, sFv-DT390 is
monovalent as compared to immunotoxins generated with full-length
bivalent antibodies. The reduced toxicity in sFv-DT390 could be
explained primarily on this affinity difference. Immunotoxins generated
with purified F(ab)` fragments of antibodies also show an in vitro loss in toxicity (generally a 1.5 log difference) when compared to
their counterparts generated with full-length antibodies (22) .
The toxicity of sFv-DT390 is comparable to that reported for
DAB
IL-2(23) . Considering using sFv-DT390 in the
clinical treatment, will there be a trade-off in evaluating a potent,
but completely neutralized toxin with one which is less potent, but not
completely blocked? A conclusive answer can only be derived from
clinical trials. However, from the in vitro data some
advantages of sFv-DT390 can be expected. First, sFv-DT390 is only
one-third of the molecular weight of UCHT1-CRM9. Therefore, sFv-DT390
can penetrate into tissue more readily. Second, in an in vitro experiment (Table 3), the same molar concentration of
sFv-DT390 and UCHT1-CRM9 was used for the serum inhibition test,
although the former is only 1/16 potent compared to the latter. The
pre-existing anti-DT antibodies in human sera could only partially
block the toxicity of sFv-DT390 while the effect of UCHT1-CRM9 was
completely blocked. Thus, sFv-DT390 could potentially bypass the
anti-DT antibodies in in vivo situations while UCHT1-CRM9
cannot. Third, sFv-DT390 contains only the variable region of UCHT1,
and should have less immunogenicity in human anti-mouse antibody
responses than the native murine antibody UCHT1. Finally, the
production cost of sFv-DT390 is much lower than that of UCHT1-CRM9.
Based on these reasons, it is conceivable that sFv-DT390, or others
with similar properties, should be useful in the treatment of T-cell
mediated diseases in humans, especially in anti-DT positive individuals
and in patients who need repeated treatments. To obtain evidence
supporting this assumption, a rhesus monkey analog of sFv-DT390 is
currently being constructed in this laboratory, and will be tested in
monkey models.
In summary, this report demonstrates that human sera can affect the toxicity and therefore the efficacy of immunotoxins generated with full-length DT mutants. It also indicates that there is a difference in anti-DT antibody repertoire between the humans and non-human primates, suggesting the immunotoxins destined for clinical trials be investigated with the appropriate serum. Furthermore, a truncated DT mutant has been suggested for generation of immunotoxins to bypass the blocking effect of pre-existing anti-DT antibodies in humans.