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.

MATERIALS AND METHODS

Bacterial and Mammalian Cells

Construction of Humanized Immunofusion Genes

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-C1 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 C2. 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.

Fermentation and Purification of Fusion Proteins

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.

Pharmacokinetic Experiments

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) .

RESULTS

Design of Gelonin Immunofusion Proteins

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 (GlySer)(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 C2 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.

Production of Fusion Proteins

Affinity of Fusion Proteins

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 nMI-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.

Activity of Fusion Proteins

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 VV::RMA::Gel (squares), VV::Gel (diamonds), and H65-RTA (circles, 11). Both donors were insensitive to H65-RTA.

Pharmacokinetics of Fusion Proteins and rGel and in Vitro Stability

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.

DISCUSSION

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; CHMC is the sequence of the SLT linker; and PSAY 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.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Xoma Corp., 1545 17th St., Santa Monica, CA 90404. Tel.: 310-829-7681; Fax: 310-828-2463.

()

The abbreviations used are: SCA, single chain antibody; RIP, ribosome-inactivating protein; rGel, recombinant gelonin; PBMC, peripheral blood mononuclear cells; SLT, shiga-like toxin; RMA, rabbit muscle aldolase; DT, diphtheria toxin; RTA, ricin A chain.

()

D. Fishwild, unpublished.

ACKNOWLEDGEMENTS

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