(Received for publication, December 30, 1994; and in revised form, April 26, 1995)
From the
B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which
the Fv portion of the B3 antibody in a single-chain form, which serves
as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety.
B3(Fv)-PE38 is specifically cytotoxic to many human cancer cell lines
and is currently evaluated in a clinical trial. Monoclonal antibodies
B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on
human carcinoma cells. The Fv regions of these antibodies were
previously cloned and expressed as the single-chain Fv-immunotoxins
B3(Fv)-PE38 and B5(Fv)-PE38, respectively. The B3(Fv)-PE38 immunotoxin
binds to antigen-positive cancer cells with a higher affinity than
B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38.
However, it is less stable and rapidly aggregates upon incubation at 37
°C. The V domains of the two Fvs are very similar,
differing by only three residues, the fourth and seventh Fr1 residues
and the fifth CDR1 residue. The V
domains of the two Fvs
vary considerably. To investigate whether any of the different V
residues may influence the stability of the B3(Fv), we
constructed a chimeric immunotoxin containing the B3V
and
the B5V
. This chimera had an improved stability and a
higher apparent antigen binding affinity and cytotoxic activity when
compared with B3(Fv)-PE38. Site-specific mutagenesis was used to show
that the V
M4L mutation has an important role in
stabilizing B3(Fv), although residues V
Ser-7 and V
Ile-28 also play a role in the increased stability. When tested
in an in vivo model system, the chimera containing the
B3V
and the B5V
had an improved antitumor
activity in a human xenograft mouse model. These studies indicate that
the common use of degenerate (``family-specific'') primers to
clone Fv fragments may introduce destabilizing mutations.
Monoclonal antibodies B3 and B5 are murine antibodies directed
against Lewis-related carbohydrate antigens, which are
abundant on the surface of many carcinomas(1) . The B3 IgG or
its fragments are currently used as the targeting moiety of
immunotoxins that are being developed as anticancer agents. Both
conventional whole IgG conjugates and single-chain recombinant
immunotoxins have been
prepared(1, 2, 3, 4) . The
single-chain Fv (
)immunotoxin of B3 is unstable at 37
°C; it undergoes inactivation mainly by aggregation, especially
upon incubation in PBS or in cell culture medium. In contrast, the
B5(Fv)-PE38 immunotoxin is less susceptible to inactivation under those
conditions, but it has lower apparent antigen binding affinity and
cytotoxicity(5) . We reasoned that we might be able to combine
the advantages of each Fv by chimerization of their variable domains,
since the Fvs of monoclonal antibody B3 and B5 bind the same
carbohydrate antigen and are homologous in sequence (particularly in
the V
domain, where 109 of 112 residues are identical).
Therefore, we sought to gain insight on the possible involvement of
individual residues of the light chain on the stability and the binding
affinity of the B3(Fv). In this paper we have characterized the
specific cytotoxicity, stability, and binding properties of
single-chain Fv immunotoxins whose Fvs are B3-B5 chimeras or B3(Fv)s
carrying point mutations in the V
domain. We found that the
chimera with B3V
and B5V
was the most stable
and potent of all the molecules tested in the in vitro assays,
and we have also compared it with the parental B3(Fv)-PE38 molecule in
an in vivo antitumor activity assay.
Figure 1:
Scheme of construction of plasmids for
expression of B3(Fv)PE38 and B3-B5 chimeric single-chain immunotoxins. L indicates the (Gly-Ser)
linker that
connects the V
to the V
in the single-chain Fv
configuration. PE38 is a truncated form of Pseudomonas exotoxin A, which lacks the toxins' own cell binding domain
I(3) . The sequences of the PCR primers B5HFr1, B5HFr4, B5LFr1,
and B5LFr4 are published(5) .
The stability of the immunotoxins in human serum at 37 °C was determined by incubation at 0.02 mg/ml in serum for 1, 2, or 4 h. Cytotoxic activities of aliquots of these immunotoxins were determined as described above and compared with the activities of the immunotoxins that were not incubated at 37 °C.
Figure 2:
Recombinant immunotoxins. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of purified immunotoxins. LaneM, molecular mass standard indicated on the left in kDa. Lane1, B3(Fv)-PE38; lane2, B3V-B5V
-PE38; lane3, B5V
-B3V
-PE38; lane4, B3(Fv)-PE38 V
M4L; lane5, B3(Fv)-PE38 V
S7T; lane6, B3(Fv)-PE38 V
M4L/S7T. 5 µg of each
protein were loaded on a 12% gel.
Figure 3:
Specific cytotoxicity of immunotoxins.
Cytotoxicity toward A431 cells was measured by the inhibition of
incorporation of [H]leucine into cell protein,
following 2 h (opensymbols) or 20 h (solidsymbols) of incubation of the cells with serial dilutions
in PBS, 0.2% bovine serum albumin of B3(Fv)-PE38 and
B3V
-B5V
-PE38 (A), or B3(Fv)-PE38
derivatives V
M4L/S7T, V
M4L, V
S7T
immunotoxins (B). Errorbars represent the
S.E. of the data.
To test the specificity of the immunotoxins, the
same cytotoxic assay was repeated on additional cell lines, which
differ in their level of B3 antigen
expression(1, 10) . As shown in Table 1,
B3(Fv)-PE38, B3V-B5V
-PE38, and B5(Fv)-PE38 had
the same spectrum of recognition of the cancer cell lines tested,
although they had different levels of specific cytotoxic activity. Also
the cytotoxic activity of each correlates with its binding affinity.
Figure 4:
Stability of immunotoxins. B3(Fv)PE38,
B3V-B5V
-PE38, and B3(Fv)PE38:V
M4L/S7T were diluted in PBS to 0.2 mg/ml and incubated at 37
°C for 1, 2, or 4 h. The molecular forms of the immunotoxins were
then analyzed by size exclusion chromatography at 4 °C as described
under ``Materials and Methods.'' The monomer peak elutes at
18-20 ml, while the aggregates elute at 11-13 ml. The
elution positions of gel filtration molecular weight standards
(Bio-Rad) are indicated above the leftuppermostchromatogram.
Fig. 5shows results from cytotoxicity assays that were
performed with aliquots of the immunotoxins that had been incubated in
PBS as 37 °C. Before treatment, B3(Fv)-PE38 had an IC of 2.2 ng/ml, and it retained 23, 10, and 5% of its cytotoxic
activity following 1, 2, and 4 h of incubation in PBS at 37 °C,
respectively. The B3V
-B5V
-PE38 chimera had an
IC
of 0.4 ng/ml, and it retained 78, 56, and 35% of its
cytotoxic activity following 1, 2, and 4 h, respectively, of incubation
in PBS at 37 °C. When incubated in human serum, B3(Fv)-PE38
retained 50, 25, and 12% of its cytotoxic activity following 1, 2, and
4 h at 37 °C, respectively, and
B3V
-B5V
-PE38 retained 80, 66, and 43% at these
time points. Table 2compares the percentage of monomeric
immunotoxin with the percentage of cytotoxic activity remaining
following incubation in PBS at 37 °C as well as the percentage of
active immunotoxin surviving after incubation in human serum at 37
°C. It demonstrates that upon incubation in PBS, the residual
cytotoxic activity correlates strongly with the relative amount of
immunotoxin monomer that survived the 37 °C incubation and that
B3V
-B5V
-PE38 is more stable than B3(Fv)-PE38
with about a 4-fold longer half-life. The inactivation rate of the
immunotoxins in human serum is slower than in PBS as we have observed
previously with other immunotoxins (11) . (
)However,
the 4:1 ratio between the half-life of the more stable
B3V
-B5V
-PE38 and the less stable B3(Fv)-PE38 is
maintained.
Figure 5:
Cytotoxic activity of immunotoxins
following incubation in PBS at 37 °C. A431 epidermoid carcinoma
cells were incubated with aliquots of the immunotoxins, which were
diluted in PBS, 0.2% bovine serum albumin following incubation at 37
°C. [H]Leucine was added 20 h after the
addition of immunotoxins.
, t = 0 hours;
, 1 h in
PBS at 37 °C; 2 h in PBS at 37 °C.
Intrinsic tryptophan fluorescence was also used to
determine the relative stability of B3(Fv)-PE38 and
B3V-B5V
-PE38 to denaturation by urea. In PBS,
both immunotoxins have an emission peak at 330 nm and a second
partially overlapping peak at 343 nm. The 330-nm peak remains unchanged
up to 2 M urea and probably reflects the emission of the PE38
part of the molecule, which requires higher urea concentration for
denaturation.
However, the peak at 343 nm increases with
urea concentration. The relative fluorescence change is shown in Fig. 6; the peak at 343 nm increased more for B3(Fv)-PE38 than
for B3V
-B5V
-PE38, indicating that the former
undergoes a conformational change, which is reflected by the
fluorescence change under a lower denaturant concentration than the
latter.
Figure 6:
Intrinsic tryptophan fluorescence of
B3(Fv)-PE38 (), B3V
-B5V
-PE38 (
),
B3(Fv)-PE38 V
M4L/S7T (large square), and
B3(Fv)-PE38 (small square) in urea. Samples of immunotoxins
were diluted to 10 µg/ml in PBS containing between 0 and 2 M urea. Fluorescence emission between 320 and 380 nm was determined
at 23 °C with excitation at 295 nm. The relative changes at 343 nm
are plotted.
Figure 7:
Alignment of B3 and B5 V amino
acid sequences. The amino acid sequence (in single-letter code)
B3V
is shown in the upperline, with
B5V
below it. Identical residues are identified by dots.
Figure 8:
Structural model of B3(Fv). The V domain is shown on the leftside of the
molecule in lightgray, and the V
domain
is shown on the rightside of the molecule in darkgray. The CDRs of both domains are
semitransparent. Residues that differ between B3V
and
B5V
are in white and are labeled according to
their positions in V
.
The yield of purified B3(Fv)-PE38 V M4L/S7T and B3(Fv)-PE38 V
M4L was 8-10%
(8-10 mg of active monomeric immunotoxin was recovered from 100
mg of recombinant protein added to the refolding buffer). The yield of
B3V
-B5V
-PE38 was also 8-10%. The yield of
B3(Fv)-PE38 and B3(Fv)-PE38 V
S7T was 2-4% (data not
shown). We have previously found that the yield of active monomeric
recombinant immunotoxin directly correlates with its stability (12) .
B3(Fv)-PE38 V
M4L/S7T,
B3(Fv)-PE38 V
M4L and B3(Fv)-PE38 V
S7T were
analyzed for stability and specific cytotoxicity just as was done with
B3(Fv)-PE38 and B3V
-B5V
-PE38.
As shown in Fig. 3B, B3(Fv)PE38 V M4L/S7T was the most
potent of the three, with IC
values of 1.2 and 0.6 ng/ml
following 2 or 20 h, respectively, of incubation on A431 cells. Its
cytotoxic activity was almost indistinguishable from that of the
chimeric B3V
-B5V
-PE38 (Fig. 3A). B3(Fv)PE38 V
M4L had IC
values of 1.8 and 1.2 ng/ml following 2 and 20 h, respectively,
of incubation on A431 cells. B3(Fv)PE38 V
S7T had the
lowest activity, with IC
values of 6.0 and 5.0 ng/ml
following 2 and 20 h, respectively, of incubation on A431 cells. Thus
the immunotoxin with V
S7T was much less potent than the
parental B3(Fv)-PE38 immunotoxin. This mutant was not analyzed further.
As shown in Fig. 4and Fig. 5and Table 2, both
the immunotoxin with the V M4L/S7T mutation and the V
M4L mutation alone aggregated in PBS at 37 °C to a lesser
extent than B3(Fv)PE38 and were also more resistant to loss of
cytotoxicity at 37 °C than B3(Fv)-PE38. B3(Fv)-PE38 VL M4L/S7T was
40, 56, and 75% aggregated following 1, 2, and 4 h in PBS at 37 °C,
respectively, and retained 55, 28, and 15% of its cytotoxic activity at
these time points. B3(Fv)-PE38 V
M4L was 55, 64, and 81%
aggregated following 1, 2, and 4 h, respectively, in PBS at 37 °C,
and retained 50, 25, and 20% of its cytotoxic activity at these time
points. When incubated in human serum, B3(Fv)-PE38 V
M4L/S7T retained 63, 38, and 33% of its cytotoxic activity
following 1, 2, and 4 h at 37 °C, respectively, and B3(Fv)-PE38
V
M4L retained 66, 45, and 33% of its activity at these
time points. When the intrinsic tryptophan fluorescence of PE38 V
M4L/S7T and of PE38 V
M4L was determined, the spectra
obtained were similar to the spectra obtained for B3(Fv)-PE38 and
B3V
-B5V
-PE38, with a peak at 330 nm that does
not change and a second peak at 343 nm that increased with increasing
urea concentration. As shown in Fig. 8, the increases at 343 nm
measured for B3(Fv)-PE38 V
M4L/S7T and B3(Fv)-PE38 V
M4L were similar to each other and were higher than the increase
observed for B3V
-B5V
-PE38 but were lower than
the increase observed for B3(Fv)-PE38, indicating that the sensitivity
of PE38 V
M4L/S7T and PE38 V
M4L to undergo a
conformational change (which is reflected by the fluorescence change)
following exposure to urea is greater than that of
B3V
-B5V
-PE38 but smaller than that of the
parental molecule B3(Fv)-PE38. Taken together, these data show that
while more stable than B3(Fv)-PE38 and having similar stabilities to
each other, both mutated immunotoxins V
M4L/S7T and V
M4L are somewhat less stable than the chimeric
B3V
-B5V
-PE38. These results further suggest
that most of the stabilizing effect of B5V
on the Fv
immunotoxin stems from its having a leucine rather than a methionine at
position 4.
Figure 9:
Blood levels of B3(Fv)-PE38 and
B3V-B5V
-PE38 in mice. Female Balb/c mice
were injected intravenously with 10 µg of immunotoxin. Mice were
bled at different times, and the immunotoxin level was measured by a
cell-killing assay, in which the ability of serum dilutions to inhibit
protein synthesis by A431 cells was tested. Results are from three mice
for each time point ± S.E.
Figure 10:
Antitumor effect of B3(Fv)-PE38 and
B3V-B5V
-PE38 in a nude mouse model. Groups of
five mice were injected subcutaneously with 3
10
cells on day 0 and were treated by intravenous injections of
B3(Fv)-PE38 (A) or B3V
-B5V
-PE38 (B) diluted in PBS containing 0.2% human serum albumin on days
4, 6, and 8 (indicated by verticalarrows) when the
tumors were established. Control mice were treated with PBS-HSA. Errorbars represent the S.E. of the data.
,
control;
, 0.1 mg/kg;
, 0.05 mg/kg;
, 0.025 mg/kg;
, 0.0125 mg/kg.
We have cloned DNA fragments encoding the variable domains of
the anticarbohydrate monoclonal antibodies B3 and B5 as single-chain Fv
immunotoxins. We employed the method of ``variable domain
shuffling'' (5) , which allowed us to obtain Fv chimeras
having V and V
segments from the two different
antibodies. The activities of the immunotoxins varied, with
B3V
-B5V
-PE38 being the most potent, with an
IC
of 0.3 ng/ml and B5V
-B3V
-PE38
being the least potent, with an IC
of 120 ng/ml following
20 h of incubation on A431 cells. The similar spectrum of recognition
of cell lines that differ in the level of B3 antigen expression (Table 1) suggests that there is no change in specificity
following chimerization of B3 and B5. We have shown previously that the
parental monoclonal antibodies recognize the same carbohydrate
antigen(5) . Since the B3V
-B5V
-PE38
chimera was the most stable and potent of the immunotoxins tested here,
it was chosen for in vivo characterization in comparison with
B3(Fv)-PE38. When tested in mice, the chimera did not differ
significantly from the B3(Fv)-PE38 immunotoxin in pharmacokinetics or
toxicity (Fig. 9, Table 3). However, it had a better
antitumor activity in a human xenograft nude mouse model (Fig. 10), where it was 2-fold more potent than B3(Fv)-PE38. The
improved antitumor activity correlates with the 3-fold difference in
cytotoxicity between the two immunotoxins when tested on A431 cells,
which were also used to establish the tumors in the mice.
The higher
cytotoxic activities (lower IC values) of the
B5V
-B3V
-PE38 chimera and the B3(Fv)-PE38
V
M4L/S7T and B3(Fv)-PE38 V
M4L mutants and
their higher apparent antigen binding affinity (not shown) may be
explained by the fact that both are more stable than the wild type
B3(Fv)-PE38. This improved stability is evident from their slower
aggregation and loss of cytotoxic activity upon incubation in PBS or in
human serum at 37 °C; very little B3(Fv)-PE38 monomer survives a
4-h-long incubation, whereas the stabilized variants (the chimera and
the mutants) retain significant cytotoxic activity. This accounts for
the fact that there is very little difference in the cytotoxic activity
of B3(Fv)-PE38 when incubated with A431 cells for 2 or 20 h, whereas
with the stabilized variants there is a 2-3-fold increase upon a
20-h incubation with A431 cells. Furthermore, the intrinsic tryptophan
fluorescence data (Fig. 6) provides additional evidence that
B3(Fv)-PE38 undergoes a conformational change (unfolding; (8) )
under less severe chaotropic conditions than the chimera or the
mutants, reflecting its inferior stability.
Site-specific
mutagenesis followed by stability and cytotoxicity assays were used to
identify which of the three V residues that differ between
B3 and B5 (Fig. 7) are responsible for the stabilizing effect.
Since the B3V
-B5V
-PE38 and B3(Fv)-PE38 V
M4L/S7T (which differs from the chimera only at the fifth CDR1
residue) had similar characteristics in the cytotoxicity and stability
assays, we conclude that the CDR residue does not play a major role in
stability. Although, judging from the differences in stability between
the B3V
-B5V
-PE38 chimera and the B3(Fv)-PE38
variants carrying B5V
residues at positions 4 and 7 or
position 4 alone, the CDR residue may also contribute to the Fv
immunotoxins' stability. Yasui et al. (13) have
recently reported that mutations in CDR residues can influence the
stability of Fvs analysis of B3(Fv)-PE38 derivatives carrying mutations
V
M4L or V
S7T separately showed that replacing
V
methionine 4 with leucine stabilized the immunotoxin
almost as much as the B3V
-B5V
-PE38 combination,
whereas replacing V
serine 7 with threonine had no
stabilizing effect and was, in fact, less active than B3(Fv)-PE38 (Fig. 3).
Examination of a structural model of B3(Fv) (Fig. 8)(10) , reveals that the side chain of V serine 7 is exposed to the solvent and does not appear to
interact directly with any other part of the molecule. The bulkier side
chain of a threonine at the same position would probably also be
exposed to solvent. V
methionine 4 is a buried residue (Fig. 7) as would probably be a leucine at the same position.
Methionine 4 does not appear to interact directly with any of the
V
residues, and the stabilizing effect resulting from its
replacement with leucine would have to be explained by an effect on the
independent folding of the V
domain. Creamer et. al.(14) have observed that there is a diminished entropy loss
when leucine replaces methionine and is folded into the buried part of
the protein. Eriksson et al.(15) have observed that
replacing leucine at buried positions of lysozyme with other
hydrophobic residues that have smaller side chains (including
methionine) destabilized the protein.Leucine is somewhat more
hydrophobic than methionine, so it may be favored in a buried
position(16, 17, 18) . In the studies cited
above, there is a small but significant difference in the stability of
the protein, and the methionine-containing variant is less stable than
the leucine-containing variant(14, 15) . The high
tendency of B3(Fv)s to aggregate may be improved by a small but
favorable alteration in its refolding thermodynamics due to V
M4L replacement.
B3(Fv)-PE38 is less stable than
B5(Fv)-PE38(5) , as were the
B3V-B5V
-PE38 chimera, B3(Fv)-PE38 V
M4L/S7T and B3(Fv)-PE38 V
M4L (not shown). It is thus
clear that part of the stability difference results from differences in
the V
domains. We have identified the B3V
residue, which is responsible for its relative instability. But
that residue cannot be mutated without a severe loss in binding
affinity, and this has important implications on the mechanism of
antigen binding by the B3 antibody. (
)Finally, we wish to
emphasize that the state of the art in cloning antibody fragments in E. coli is the use of ``family-specific primers''
for the PCR amplification of the variable region segments from
cDNA(19, 20, 21, 22) . Our data
demonstrates that one must be cautious when such techniques are used,
because the N termini of the obtained clones may not match the original
protein sequence, and this alteration can lead to Fvs with altered
stabilities and affinities.