(Received for publication, September 26, 1994; and in revised form, March 10, 1995)
From the
Fusion proteins between cell-targeting domains and cytotoxic
proteins should be particularly effective therapeutic reagents. We
constructed a family of immunofusion proteins linking humanized Fab,
F(ab`), or single chain antibody forms of the H65 antibody
(which recognizes the CD5 antigen on the surface of human T cells) with
the plant ribosome-inactivating protein gelonin. We reasoned that such
an immunofusion would kill human target cells as efficiently as the
previously described chemical conjugates of H65 and gelonin (Better M.,
Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A.
H. C., and Carroll, S. F.(1994) J. Biol. Chem. 269,
9644-9650) if both the recognition and catalytic domains remained
active, and a proper linkage between domains could be found.
Immunofusion proteins were produced in Escherichia coli as
secreted proteins and were recovered directly from the bacterial
culture supernatant in an active form. All of the immunofusion proteins
were purified by a common process and were tested for cytotoxicity
toward antigen-positive human cells. A 20-60-fold range of
cytotoxic activity was seen among the fusion family members, and
several fusion proteins were identified which are approximately as
active as effective chemical conjugates. Based on these constructs,
immunofusion avidity and potency can be controlled by appropriate
selection of antibody domains and ribosome-inactivating protein.
The antigen-binding domains of antibodies are ideal delivery
agents for cytotoxic compounds to the surface of cells, and
immunoconjugates consisting of whole antibody or antibody domains
linked to proteins that disrupt cellular protein synthesis have been
widely described. Immunoconjugates have typically been linked in
vitro from antibodies and cytotoxic proteins with
heterobifunctional cross-linking agents. Recent advances in antibody
engineering, however, now make it possible to express a variety of
antibody domains independently in microorganisms, and to express
antibody domains as fusion proteins with a variety of enzymes. Several
examples of single chain antibody (SCA)(
)and Fab
fusion proteins to cytotoxic enzymes have been described (1-3).
The best characterized fusion proteins are those between SCA and Pseudomonas exotoxin A(4, 5, 6, 7, 8) . The fusion proteins are often as cytotoxic or more so toward antigen-positive target cells as the chemical conjugates between antibody and enzyme. These fusion proteins are typically produced in Escherichia coli as insoluble inclusion bodies and refolded in vitro to an active from. Although recovery and refolding yields can approach 0.05 g/liter, the production process can be complex(9) .
We
recently identified immunoconjugates between bacterially produced
antibody domains and the plant ribosome-inactivating protein (RIP)
gelonin that are extremely effective at killing antigen-positive target
cells(10) . In this case, the antibody domains derived from the
murine H65 antibody recognize the CD5 antigen on the surface of mature
human T cells and a subpopulation of B cells(11) . Although the
antigen-binding domain and recombinant gelonin (rGel) were both
secreted as fully active, properly folded protein from E. coli at yields approaching 1 g/liter(12, 13) , assembly
of the active molecule required in vitro conjugation. Since
both components were expressed efficiently as separate chains, we
generated fusion proteins between gelonin and the humanized
antigen-binding domains Fab, F(ab`), and SCA and expressed
them as secreted proteins as well. Our previous studies indicated that
the exact positioning of the two functional units in a chemical
conjugate greatly influenced activity(10) . As a result, we have
now constructed a family of similar fusion proteins to identify optimal
domain arrangements. Each family member was expressed as a secreted
protein in E. coli and purified from the culture broth in an
active form. A range of cytotoxic activities was seen among the fusion
family members. Several fusion proteins were identified whose
activities approached the most effective chemical conjugates.
The gelonin gene was fused in-frame to each SCA at either the 5`- or 3`-end with nucleotides encoding linker polypeptides derived from shiga-like toxin (18, SLT) or rabbit muscle aldolase (19, RMA) positioned between the gelonin and SCA domains. Direct fusions of SCA genes to gelonin without the SLT or RMA encoding linker were also constructed.
The gelonin gene was similarly linked
to the 5`-end of V-J-C or V-J-C
1 encoding
sequences, and the fusion genes were assembled into a dicistronic
message with the cognate Fd or
constant region-encoding sequence,
respectively. Direct fusions between gelonin and antibody were
prepared, as were fusions encoding the RMA or SLT linker. Fab fusions
with two gelonin genes were constructed as well by inclusion of a
gelonin-
and gelonin-Fd gene into a single operon. The
gelonin::RMA::
fusion gene was also incorporated into a
dicistronic message with an Fd` gene which encoded both IgG1 interchain
hinge cysteine residues and the first 9 amino acids of C
2.
Inclusion of this segment allows direct E. coli expression of
the divalent F(ab`)
,(13) , and
F(ab`)
-fusion protein was produced. The amino acid
sequences at the junctions between gelonin and antigen-binding domains
are shown in Table I. The DNA sequence at gene segment junctions
were verified with the Sequenase Version 2.0 DNA Sequencing Kit or
TAQuenase Cycle Sequencing Kit (U. S. Biochemical Corp.), as were the
DNA sequences of all genes assembled from polymerase chain
reaction-generated DNA fragments.
The cells were separated from the culture
supernatant (which contains the recombinant protein) with a 0.2-µm
Microgon Hollow Fiber cartridge (1.0 m). The cell-free
fermentation broth (approximately 7 liters) was concentrated and
diafiltered with 20 liters of 10 mM sodium phosphate, pH 7.0,
using a DC10 with an S10Y10 Amicon cartridge to a final volume of
approximately 3 liters.
The concentrated culture supernatant (in 10
mM sodium phosphate, pH 7.0) was applied to a CM-Spherodex
column and the fusion protein was eluted with 300 mM NaCl.
Fractions containing fusion protein were applied to a phenyl-Sepharose
Fast Flow (Fab fusions) or butyl-Sepharose Fast Flow resin (SCA
fusions) in 1.5 M (NH)
SO
,
0.15 M NaCl and 20 mM HEPES, pH 7.0. The fusion
protein was eluted with 20 mM HEPES, concentrated, and applied
to a Sephacryl 200 gel filtration column equilibrated in
phosphate-buffered saline. The purified immunofusion protein was stored
at -20 °C in phosphate-buffered saline.
Plasma concentrations of immunofusion protein and gelonin were determined by enzyme-linked immunosorbent assay. To detect immunofusion protein, recombinant soluble CD5 (Xoma Corp.) was the capture reagent; to detect gelonin, affinity-purified rabbit anti-gelonin was the capture reagent. Biotin-labeled, affinity-purified rabbit anti-gelonin (Xoma Corp.) was used as the signal detecting reagent with alkaline phosphatase-labeled streptavidin (Zymed Laboratories Inc., San Francisco, CA) and p-nitrophenylphosphate.
A two-compartment pharmacokinetic equation was used to describe the change in concentration with time. The data were fitted by weighted nonlinear least squares analysis, using the software program PCNONLIN (Statistical Consultants, Inc., Lexington, KY). The clearance rate (CL, ml/min/kg) was calculated from the primary curve fit parameters as described(21) .
Table Ischematically illustrates the immunofusion proteins we
produced and shows the amino acid sequence at the fusion junctions.
Gelonin was positioned at either the N- or C terminus of the fusion
protein. The SCA fusions were constructed to encode either the light
chain or heavy chain variable region (V or V
,
respectively) at the N terminus of the antigen-binding domain with a
15-amino-acid flexible peptide linker (Gly
Ser)
(17) between the variable domains. A divalent F(ab`)
fusion protein (two Fab` units and two gelonin domains) was
engineered by introducing the entire human IgG1 hinge region and nine
amino acids of the C
2 domain, as described(13) . In
this case, both the monovalent Fab` fusion and divalent form could be
recovered from the bacterial culture and tested separately for
activity.
We also engineered possible intracellular release mechanisms into fusion proteins by introducing one of two short peptide sequences between the antigen targeting domain of the he3 H65 antibody and gelonin. These peptide segments from the E. coli shiga-like toxin (18) and rabbit muscle aldolase (19) are 20 amino acids in length. The SLT sequence, CHHHASRVARMASDEFPSMC, contains a disulfide-bounded peptide with a recognition site for trypsin-like proteases and resembles the cleavable disulfide loop of Pseudomonas exotoxin A and diphtheria toxin (DT), while the RMA sequence, PSGQAGAAASESLFISNHAY, contains several sites that are susceptible to the lysosomal enzymes Cathepsin B and Cathepsin D(19) . We reasoned that these peptides were likely to be cleaved intracellularly resulting in gelonin release. Several direct fusions without either the SLT or RMA linker peptides were also constructed.
Figure 1:
Competitive binding assay with he3 H65
IgG, Fab, SCA, and gelonin immunofusion proteins. A,
comparison of IgG and Fab to immunofusions. Results from binding
experiments were analyzed by the weighted non-linear least-squares
curve fitting program (MacLigand), adapted from the Ligand program (35)
which assumes that all competing molecules are capable of binding to
antigen. Objective statistical criteria, including the F-test and the
extra sum of squares principle, were used to evaluate goodness of fit
and to discriminate between models. Nonspecific binding was treated as
a parameter subject to error and was fitted simultaneously with other
parameters. B, comparison of SCA to SCA-immunofusion protein.
MOLT-4M cells (3
10
cells/well,) were
incubated at 4 °C for 5 h with 0.001-100 nM
unlabeled blocking agents in the presence of 0.1 nM
I-labeled he3 H65 IgG. Cells were washed three times
and 100 µl of 2 N NaOH was added to each well to
solubilize the cells. Extracts were counted in a Beckman Gamma 8000
gamma counter.
I-Labeled he3 H65 IgG was prepared using
20 µg of purified IgG with lactoperoxidase beads (Enzymobeads,
Bio-Rad) in the presence of 1-2 mCi of
I (Amersham,
IMS-30) as described by Bio-Rad. The labeled he3 H65 IgG was purified
on a Sephadex G-25 column.
Table II highlights
the activity of fusion proteins on HSB2 cells. As expected, some fusion
proteins were more cytotoxic than others. In general, fusions
containing the SLT linker were more cytotoxic than fusions containing
the RMA linker or no linker at all. Fusions containing the SCA at
either the N or C terminus of the molecule were equally effective at
killing cells. There also did not seem to be a clear advantage to
fusion proteins containing Fab or SCA. A striking difference was seen,
however, between monovalent and divalent forms of the fusion proteins.
The (Gel::RMA::, Fd`)
molecule was roughly
10-20-fold more effective at cell killing than the monovalent
form. Interestingly, the Gel::SLT::
, Gel::SLT::Fd molecule, a Fab
with two gelonin moieties, was more cytotoxic than a Fab fusion linked
to gelonin on either
or Fd. In contrast, Gel::RMA::
,
Gel::RMA::Fd was more active than the Gel::RMA::
,Fd fusion
protein, but only as active as the Gel::RMA::Fd,
fusion protein.
Different patterns of cytotoxicity emerged from the assays with
PBMC. In a comparison among SCA, Fab, and F(ab`) fusions
with a single linker, the divalent immunofusion was clearly the most
potent (Table III). The Fab conjugates were somewhat more active
than the single chain fusions, although in this assay, an IC
variation of less than 2-fold is unlikely to be meaningful. As
seen on HSB2 cells, the Fab fusion with two gelonin domains
(Gel::RMA::
, Gel::RMA::Fd) was about as potent as immunofusion
protein with a single gelonin.
The role of a cleavable peptide linker between functional domains is illustrated in Table IV. In general, the introduced linkers made little difference, although for gelonin fusion to the N terminus of SCA, inclusion of either RMA or SLT increased activity by about 3-fold. The immunofusions without a specific linker may contain an amino acid sequence at the interdomain junction that creates a susceptible cleavage site or alternatively, these gelonin immunofusion proteins may be transported to the cytoplasm of the cells intact and remain in an active form. Since in some cases a cleavable linker enhances activity, separation of gelonin and binding domain may be optimal. Another interesting observation is that with the SCA and Fab fusions to the C terminus of gelonin that include RMA, linkage through the heavy chain gives more effective fusions than does linkage through the light chain.
Another relevant measure of reagent potency, especially for low molecular mass immunofusion proteins which clear rapidly in animals (see below), is how long they must be in contact with target cells in order to cause maximal cytotoxicity. As shown in Fig. 2, two immunofusion proteins with SCA approach maximal cytotoxicity quickly. Thus, as was observed with gelonin chemical immunoconjugates (22), a brief contact time is sufficient for gelonin immunofusion proteins to achieve maximal cytotoxicity. As shown, this is true for fusions both with or without the RMA linker. Cells from both donors were insensitive to the ricin A chain (RTA) chemical conjugate to H65 (H65-RTA, 11), again highlighting our observations that targeted gelonin in particular shows improved potency and efficiency(10, 22) .
Figure 2:
Effect of exposure time on immunofusion
protein potency. At the indicated times, PBMC were washed to remove
unbound immunofusion protein and then incubated in medium for up to a
total of 90 h. Cytotoxicity was determined as described (13, 22).
Results from two different donors are displayed in panels A and B as ICversus exposure time.
Shown are V
V
::RMA::Gel (squares),
V
V
::Gel (diamonds), and H65-RTA (circles, 11). Both donors were insensitive to
H65-RTA.
Figure 3: Plasma clearance of rGel or fusion proteins in rats. rGel or fusion protein was administered at a dose of 0.1 mg/kg in male CD rats. Symbols represent mean plasma concentrations (± SE, n = 3). The lines accompanying the data points represent curve fits to the data.
In an in vitro assay of fusion
protein stability, we incubated SCA, Fab, and F(ab`) fusion
proteins in 90-95% normal human serum for up to 24 h, and
aliquots were removed and assayed for cytotoxicity against HSB2 cells.
Under these conditions, less than 30% of the activity was lost over the
24-h period (data not shown). From first-order rate plots of activity versus time, we estimated functional half-lives in human serum
at 37 °C of 43, 63, and 347 h for the SCA, Fab, and F(ab`)
fusions, respectively.
Multifunctional fusion proteins consisting of enzymes and targeting domains should have many pharmaceutical and diagnostic applications. Two important considerations, however, are how efficiently these molecules fold after expression (yield) and how much of the individual domain function is retained (activity). Recently, several examples of fusion proteins to antibody domains have been described, including fusion to plasminogen activators(23) , alkaline phosphatase(24) , protein A(25) , and bacterial toxins such as Clostridium perfringens toxin(2) , Pseudomonas exotoxin (PE; 4-8), and diphtheria toxin (DT; 26). In these examples the fusion proteins were expressed in bacteria, and the recombinant proteins were purified either directly as a secreted protein (2) or more often after refolding from intracellularly expressed protein. In several examples, the fusion proteins retained the activity of each functional domain and were of equal or superior activity to the chemical conjugates between domains.
Genetic fusions of targeting domains to cytotoxic proteins such as Pseudomonas exotoxin A and DT may be particularly effective because the toxins themselves contain disulfidebounded internal peptide sequences that are substrates for intracellular proteases and may be cleaved concurrent with release and translocation of the catalytic domain into the cytoplasm(27) . The importance of a labile, disulfide-bounded loop for intracellular delivery of a catalytic domain was highlighted by O'Hare et al.(28) , who engineered the short amino acid sequence from DT containing the protease-sensitive and disulfide-bounded loop into a fusion protein between the A chain of the type II RIP ricin (RTA) and staphylococcal protein A. Only the fusion protein with the proteolytically nicked DT segment was cytotoxic to immunoglobulin-coated cells, suggesting that only when RTA is released in the cytosol did it form an effective fusion protein. Thus, when the proper signals were included in the fusion protein, the desired biological functions could be reconstituted.
Since gelonin and RTA share a common catalytic
mechanism and are structurally related proteins, we included the SLT or
RMA linker between the antigen binding and catalytic domains. We
reasoned that introduction of a specific intracellular cleavage
mechanism such as that found in Pseudomonas exotoxin A and DT
fusion proteins might be necessary for maximal cytotoxicity of gelonin
fusion proteins. Our data suggest, however, that gelonin fusions with
an engineered protease-sensitive linker are often no more cytotoxic to
human PBMC than those without such a linker. We subsequently observed
that immunotoxins prepared by coupling gelonin to H65 antibody domains
via a non-reducible thioether linkage retained potent activity against
human PBMC.(
)In addition, similar findings were
recently reported with gelonin conjugates to another
antibody(29) . Perhaps unlike the type II RIP ricin, the type I
RIP gelonin does not require specific separation from any delivery
agent for intracellular activity. Additional experiments will be
required to clarify this point since a fusion protein between the type
I RIP saporin and basic fibroblast growth factor (30) apparently
requires intracellular proteolysis for activation of RIP activity.
We were interested in identifying reagents which could specifically kill T cells implicated in human disease. We reasoned that the most useful molecules would be those which are produced efficiently and exhibit the highest degree of specific cytotoxicity with the lowest inherent toxicity. Previous work with chemically linked immunoconjugates has demonstrated that no particular RIP is likely to form the most effective conjugate with all cell-targeting molecules, and we found that the most cytotoxic conjugates with the anti-human CD5 antibody H65 are those with gelonin(10) . Since both the he3 H65 antigen-binding domains and gelonin are expressed in E. coli at high yield, we explored whether fusion proteins between these molecules could be expressed in E. coli as well.
The molecules described here exhibit a range of cytotoxic activity that varies >60-fold on HSB2 cells and > 20-fold on PBMC. The divalent immunofusions are about as active as the most effective chemical conjugates between antigen-binding domains and gelonin (compare to 10). Some of the monovalent Fab and SCA fusion proteins are also very cytotoxic to PBMC and are as potent as the H65-RTA immunoconjugate that has been tested clinically(31, 32) . The range of potencies seen among these immunofusion proteins may not be unexpected, since individual members of the immunofusion family differ in antigen affinity, and the orientation of constituent domains (antigen binding, catalytic, and linker) can affect activity.
The targeted cytotoxic
molecules described here are cleared rapidly in vivo in rats
with an inverse correlation between clearance rate and molecular mass.
The clearance rate for the Fab-gelonin fusion protein, however, is
similar to that of chemically linked immunoconjugates between Fab and
gelonin(10) . Since both immunofusion proteins and the similarly
sized immunoconjugate clear rapidly, they must target and kill cells
quickly if they are to be clinically effective. Three lines of evidence
suggest that they can be effective. The fusion proteins described here
remain intact in vivo and do not lose activity even after
prolonged incubation in vitro in human serum. In addition,
SCA-fusion proteins approach their maximal cytotoxicity on human PBMC
rapidly. This short contact time is much less than that required with
whole antibody ricin immunoconjugates, for example, in a similar
assay(22) . Furthermore, Fab and F(ab`) chemical
conjugates to gelonin can deplete human T cells efficiently in vivo in a human peripheral blood lymphocyte-reconstituted severe
combined immunodeficient mouse model(32) . Because the
immunofusion proteins described here and the chemical conjugates tested
in severe combined immunodeficient mice have similar activity in
vitro (within 5-fold) and similar in vivo clearance, we
expect that the fusion proteins would also eliminate human T cells in
the severe combined immunodeficient mouse model. Further in vivo testing of the immunofusion proteins appears warranted.
The
most important conclusions from the work described here are that
several H65-gelonin immunofusion proteins are as cytotoxic to human
PBMC as the H65-RTA immunoconjugate which has seen wide clinical use,
and one protein (Gel::RMA::, Fd`)
is as cytotoxic as
the most effective chemical immunoconjugates between H65
antigen-binding domains and recombinant gelonin. Importantly, these
fusion proteins can be prepared directly from the supernatant of
induced E. coli cultures. These findings are directly relevant
to the clinical potential of these fusion protein products.
Shown are the fusion proteins and the fusion junction amino acid
sequences. The columns Syn illustrate amino acids that were introduced
to allow gene cloning. PK are the carboxyl-terminal residues of
gelonin; SS are the carboxyl-terminal residues of V; DI are
the carboxyl-terminal residues of
; EI are the amino-terminal
residues of Fd; G is the amino-terminal residue of gelonin;
CH
MC is the sequence of the SLT linker; and
PS
AY is the sequence of
RMA.
Cytotoxicity assays with the HSB2 T cell line were performed as
described (13, 34). By comparison with an untreated control, the
concentration of immunofusion that results in 50% inhibition of protein
synthesis (IC) was calculated.
Cytoxicity assays with PBMC were performed as described (13, 34). No. represents the number of times the assay was repeated on different PBMC samples.
Cytotoxicity assays with PBMC were performed as described in Table III.