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The Journal of Immunology, 2000, 165: 7050-7057.
Copyright © 2000 by The American Association of Immunologists

Fab Chains As an Efficient Heterodimerization Scaffold for the Production of Recombinant Bispecific and Trispecific Antibody Derivatives1

Reinhilde Schoonjans, An Willems, Steve Schoonooghe, Walter Fiers, Johan Grooten2 and Nico Mertens

Molecular Immunology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow’s medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75–100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)2 ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bispecific Abs (BsAbs)3 are versatile tools in the development of new experimental therapies of various diseases. Typically, one part of the BsAb specifically recognizes a target molecule or cell (e.g., a cancer cell), whereas the other part is directed to an enzyme, toxin, virus, or immune effector cell. The production of BsAbs is usually achieved by chemical cross-linking of Fab (1) or by the hybrid hybridoma technique (2). The requirement for extensive postproduction purification steps to isolate the bispecific component from other Ig-derived by-products has driven the application of recombinant DNA methodology and Ab engineering techniques for production of BsAbs in bacterial expression systems. Bispecific single-chain variable fragment (scFv)2 heterodimers have been produced by direct genetic fusion (3), or indirectly by fusion to helical heterodimerization domains (4, 5, 6). To avoid the rapid blood and whole-body clearance exhibited by these small-sized molecules (7, 8), strategies have been developed to produce intermediate- and larger-sized recombinant BsAb. Efficient heterodimerization of complete heavy chains was accomplished by engineering complementarity into the domain interfaces of two CH3 molecules (9). For optimal results, also a disulfide bridge had to be introduced into the molecule to stabilize the heterodimer (10). Also single Ab domains, such as CL (11) or CH3 (12), have been applied for homodimerization, generating bivalent minibodies. These intermediate-sized molecules avoid clearance in the kidney, whereas they still have a more efficient tissue penetration than complete Abs, which makes them better suited for in vivo therapeutic application (12). To transform these homodimeric bivalent molecules into heterodimeric BsAbs, spontaneous interaction between the complementary single domains CL and its natural partner CH1 was applied (13). This approach generated a stabilized heterodimer due to the presence of a natural disulfide bridge and gave rise to 63% of heterodimerization when both domains were coexpressed in Escherichia coli (13).

In contrast to E. coli, mammalian cells possess extensive control mechanisms that prevent incorrectly folded proteins to proceed along the secretory pathway (14). Consequently, heterodimerization of CL and CH1 domains might be further enhanced in mammalian cells. To verify this possibility, we analyzed the efficiency of heterodimerization of CL and CH1 domains coexpressed in mammalian cells. The results show that additional VL:VH contribution to CL:CH1-mediated heterodimerization greatly enhances efficient heterodimerization and secretion. Fusion of a scFv molecule to one or both of the VL-CL (L) and VH-CH1 (Fd) chains resulted in nearly exclusive secretion of heterodimeric fusion molecules, viz BsAbs or trispecific Abs (TsAbs) of intermediate size with fully functional bi- or trispecific activities. These results put forward the L:Fd interaction as an efficient heterodimerization scaffold for the generation of multifunctional Ab derivatives in mammalian cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

HEK293T, a human embryonic kidney cell line transfected with SV40 large T-Ag (SV40TtsA1609) (15), was used for transient eukaryotic expression. TE2 cells are murine and CD3+ Th1-type cells are T cells (16). MO4I4 cells are C3H mouse-derived MO4 fibrosarcoma cells transfected with the human placental alkaline phosphatase (hPLAP) gene (17, 18). BALB/c-derived myeloma BCL1 expresses surface IgM{lambda} (19).

Plasmids and gene assembly

Restriction enzymes, DNA modifying enzymes, and DNA polymerase were used as recommended by the manufacturers. DNA amplification was performed with Vent-DNA polymerase (New England Biolabs, Beverly, MA). E6 and 2C11 denote the genes or gene fragments of an anti-hPLAP and an anti-murine TCR-associated CD3{epsilon}-chain (anti-CD3) mAb, respectively. Expression plasmids were constructed in pCAGGS (20). Cloning of the L chain of E6 mAb anti-hPLAP (IgG2b/{kappa}) in the vector pSV51E6L has been described previously (21). The E6Fd fragment encodes VH, CH1, and the first five amino acids EPSGP of the upper hinge region. Gene assembly was conducted by introduction of suitable restriction sites using modifying PCR primers. All PCR-derived fragments were sequence verified after cloning. Fusions with E6CH1 or E6CL include the "elbow" regions (EMKRAD and SAAKTT from the L and Fd chains, respectively) of Fab chains. The isolated coding sequence for the CL domain was fused to the signal sequence of the E6 heavy chain. scFv(anti-BCL1) and (G4S)3-scFv(anti-CD3)-(His)6 were amplified from pQE-bssFvB1–2C11 (22) and genetically fused to the C terminus of CL or CH1 via a DVPSGPG or (G4S)3 linker, respectively.

Production of Ab fragments

For transient expression, HEK293T cells were transfected according to the Ca3(PO4)2 precipitation method (23). Twenty hours before transfection, HEK293T cells were seeded at 4 x 106 cells/175 cm2. Fourteen micrograms DNA of each expression plasmid was added to the cells for 24 h, after which the cells were covered with supplemented DMEM containing 5 mg/L bovine insulin, 5 mg/L transferrin, and 5 µg/L selenium (ITS) replacing FCS. Medium was harvested every 48 h after transfection. Gel filtration was performed on an XK 16/88 Superdex 200 column (Amersham Pharmacia Biotech, Piscataway, NJ) calibrated with a commercial protein standard mix (Bio-Rad, Richmond, CA). A sample volume of 1 ml was loaded and the column was developed in 15 mM NaH2PO4, 150 mM NaCl, pH 7.5, at 1 ml/min. The concentration of the produced recombinant Ab fragments was determined on Western blot; samples were applied in three different dilutions and compared with a serial dilution of a standard of purified TsAb of known concentration.

Western blotting, immunodetection, and densitometry

Medium fractions (50x concentrated) of transfected cells, corresponding to 1 ml supernatant, were diluted with nonreducing SDS sample buffer, boiled for 5 min, fractionated by 10% SDS-PAGE, and blotted to a nitrocellulose membrane. Subsequent functional detection of anti-hPLAP activity was achieved directly by incubation of the membrane with hPLAP (Sigma, St. Louis, MO). Immunodetection of the proteins on blot was as described previously (21); goat anti-mouse Ig {gamma}{kappa} serum (Sera-Lab, Crawley Down, Sussex, U.K), anti-E-tag (Amersham Pharmacia Biotech, Rainham, U.K.), anti-goat IgG serum conjugated to alkaline phosphatase, and anti-mouse IgG1 conjugated to alkaline phosphatase were used. For densitometric measurements, blots containing immunoreactive signals were scanned and analyzed with ImageMaster VDS software (Amersham Pharmacia Biotech).

Cellular binding

Flow cytometry was performed by concentrating cell culture supernatant containing Fab-scFv BsAb or Fab-(scFv)2 TsAb and dialyzing to PBS, after which it was supplemented with 0.5% BSA and 0.02% azide. Washed cells (2 x 105) were then resuspended in 100 µl of concentrated and dialysed BsAb, TsAb, E6 mAb, or B1 mAb (5 µg/ml), after which cells were incubated for 30 min at 4°C. Following three wash procedures, the cells were incubated for 30 min with 1 µg/ml of fluorescein-conjugated goat anti-mouse Fab (Organon Teknika, Durham, NC). After a final wash procedure, cells were analyzed by FACS. Binding of 145-2C11 mAb was detected directly with an anti-hamster FITC-coupled antiserum (Sera-Lab).

Cellular binding (cellular ELISA) was performed by preincubating 106 TE2 cells with 1 µg Fc-Block (PharMingen, San Diego, CA) for 30 min. Two micrograms mAb E6 or 2 µg concentrated and dialyzed Fab-(scFv)2 TsAb were added to 2 x 105 washed TE2 cells in 100 µl PBS, supplemented with 0.5% BSA and 0.02% azide, and were incubated for 30 min at 4°C. After three wash procedures with supplemented PBS, the cells were incubated for 30 min with 5 U/ml hPLAP (Sigma). After a final wash procedure, cells were resuspended in 600 µl 10% DEAE with p-nitrophenyl phosphate. Two hundred microliters of each sample was transferred in triplicate to a multiwell plate, after which A405 was measured.

Absorbing-out of Ab with cells was performed by incubating three times with 6 x 105 target cells (MO4I4, BCL1, or TE2) or irrelevant cells (SP2/0). The nonbound fraction was analyzed on Western blot and revealed with anti-mouse IgG serum.

Surface plasmon resonance

Affinity analysis was performed using a BIAcore 2000 (BIAcore, Uppsala, Sweden). Fab were prepared by papain digestion and subsequent protein A chromatography (Pierce, Rockford, IL). His-tagged TsAb was purified from serum with immobilized metal affinity chromatography using cobalt as a ligand. The TsAb as well as hPLAP (Sigma) were further purified on phenyl Sepharose and Q-Sepharose columns (Amersham Pharmacia Biotech).

T cell proliferation assay

Splenocytes from syngenic C3H/He mice and BALB/c mice were used for MO4I4 fibrosarcoma cells and BCL1 lymphoma cells, respectively. All mice were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). MO4I4 and BCL1 tumor cells were pretreated with 50 µg/ml mitomycin C at 37°C in the dark for 12 h or 90 min, respectively. After removal of mitomycin C, 5 x 104 treated cells were cocultured with 1 x 105 splenocytes in a round-bottom well in the presence of 0.5 µg/ml of bispecific Fab-scFv or trispecific Fab-(scFv)2 molecule. After 48 h, the culture was pulsed with 0.5 µCi of [3H]thymidine (1 mCi/ml). After 18 h the cells were disrupted by freeze-thawing; the DNA was spotted on a filter and washed. The incorporated radioactivity was measured by scintillation counting (Top-Count; Packard, Meriden, CT). All experiments were performed in triplicate.

51Cr release assay

Redirecting cytotoxicity from CTL responses was assayed using a standard 51Cr release assay with syngenic CTL cells that were alloreactively primed. Briefly, 4 x 106 splenic responder cells (C3H/HeOUico) were mixed with 4 x 106 splenic stimulator cells (C57BL/6) treated with 50 µg/ml mitomycin C for 60 min at 37°C in the dark. The mixed cell population was cocultured in 2-ml cultures in supplemented RPMI 1640 in the presence of 30 U/ml of murine IL-2. These cultures were incubated at 37°C in 7% CO2 in humidified air for 5 days.

MO4I4 cells were incubated with 150 µCi Na51CrO4 (Amersham Pharmacia Biotech) for 90 min at 37°C and washed carefully to minimize spontaneous release. Effector cells from the mixed lymphocyte culture were harvested and washed; 2.5 x 105 cells were plated in triplicate in 96-well U-bottom plates containing 5 x 103 tumor cells (E:T ratio 50:1) and Fab-scFv BsAb (1 µg/ml) in a total volume of 200 µl. After a 4-h incubation at 37°C, 30 µl of the culture supernatant was transferred to a Lumaplate (Packard), air dried, and counted. The percentage of specific lysis was calculated as 100 x (experimental release) - (spontaneous release)/(maximum release) - (spontaneous release). Maximum release was the value obtained from target cells incubated with 2% SDS. Spontaneous release never exceeded 14% of the maximum release.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CL:CH1 heterodimerization is inefficient in mammalian cells unless enlarged with variable domains

To assess the eukaryotic secretion of homo- and heterodimers from individual domains of Ab L and Fd chains, HEK293T cells were transiently (co)transfected with pCAGGS expression vectors containing as an insert the isolated CL or CH1 domain. These domains are derived from mouse Ab E6 (IgG2b,{kappa}) (24) specific for hPLAP (25). However, no heterodimeric product could be detected, even not if for the purpose of more sensitive detection the CH1 domain was modified with an E-tag.

To assess whether the presence of either the VH or the VL domains is required for progression of these Ab derivatives through the endoplasmic reticulum, the CL and CH1 domains were coexpressed with their corresponding extended counterparts, namely, the complete Fd chain and the native L chain, respectively (i.e., CL:VHCH1 and VLCL:CH1). Also here, no secreted heterodimers, either CL:Fd or L:CH1, could be detected. Only L monomers and L:L homodimers were demonstrated in culture fluids of L gene-(co)transfected HEK293T cells. However, coexpression of CL and CH1, both enlarged with their corresponding variable domains (in fact representing L and Fd chains) generated efficient expression of L:Fd heterodimers (Fab). The Fd chain on its own was never detectable, neither as a monomer nor as a homodimer. Thus the Fd chain can only be secreted in the form of a heterodimer with the L chain, whereas the L chain preferentially forms heterodimers with the Fd chain upon coexpression. These results identify the L and Fd chains as a minimal configuration for obtaining efficient heterodimerization and secretion of CL:CH1-containing Ab derivatives in mammalian cells.

Fab-constituting L and Fd chains mediate efficient heterodimerization of scFv molecules

Because enlarging the CL and CH1 domains with VL and VH significantly increases the efficiency of heterodimerization and secretion, we wanted to establish whether the corresponding L and Fd chains could be used as a heterodimerization scaffold to produce BsAbs and even TsAbs. Because the L:Fd heterodimer itself constitutes a functional binding site, a C-terminal elongation of the L and Fd chain with a peptide linker and scFv molecule(s) would generate BsAb or TsAb with broad action radius. To create a model molecule, a scFv specific for the murine myeloma BCL1 Id (22) was fused to the C terminus of the L chain with a six-amino-acid linker, whereas the anti-murine CD3{epsilon} scFv (22) was fused to the C terminus of the Fd chain with a (Gly4Ser)3 linker.

Coexpression of the fusion genes (L-scFv and Fd-scFv) with the native complementary chain (L or Fd) is then expected to yield L:Fd-scFv and L-scFv:Fd heterodimers as schematically represented in Fig. 1Go, A and B. Western blot analysis of the culture supernatant of HEK cells, cotransfected as described above, showed secretion of the expected heterodimers (Fig. 1Go, D and E). The secreted Ab products consist predominantly of the L-scFv:Fd and L:Fd-scFv heterodimers, along with minor bands representing L-scFv or native L chain monomers and homodimers.



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  		FIGURE 1. Heterodimerization of scFv molecules by L and Fd chains. Schematic representation of a BsAb created by extension of the C terminus of the L chain (A) or the Fd chain (B), or of a TsAb by extension of both the L and Fd chains with a scFv (C). Secretion of L:Fd heterodimers by cotransfected HEK293T cells was assayed by Western blotting and immunodetection with anti-murine IgG {gamma}{kappa} anti-serum or with hPLAP. D, L chain elongated with scFv. E, Fd chain elongated with scFv. F, elongation of both L and Fd chains. Darker ellipses, CL-containing fusion molecules; brighter ellipses, CH1-containing fusion molecules. Arrowheads show respective positions on the blot. M, Molecular mass markers (kDa).

 
Similarly, coexpression of both enlarged L-scFv and Fd-scFv chains in HEK293T cells yielded a dominant L-scFv:Fd-scFv heterodimer. This was concluded based on the observed and expected 100 kDa of the dominant protein band (Fig. 1Go, C and F). Staining of the blotted proteins with hPLAP to reveal the specificity of the Fab portion (which is only formed in the heterodimer) confirmed the position of BsAbs and TsAb (Fig. 1Go, D–F). The presence of a stabilizing disulfide bridge in Fab-scFv and Fab-(scFv)2 heterodimers was confirmed by SDS-PAGE under reducing conditions (results not shown).

The relative amount of the secreted products was estimated by densitometric scanning of immunoreactive signals on a Western blot developed with anti-murine IgG {gamma}{kappa} serum at different sample dilutions. Up to 90% of the secreted Ig-derived proteins was in the correct heterodimeric format when either Fd-scFv({alpha}-CD3) was heterodimerized with the native L chain or L-scFv({alpha}-BCL1) was heterodimerized with the Fd chain or with the L-scFv fusion product. Sometimes, an excess of L chain-derived proteins was observed, which were either in a monomeric form or appeared as a disulfide-stabilized dimer. However, Fd chain derivatives always appeared as a heterodimer. Expression levels of the heterodimeric products were estimated by calibrated immunostaining to be 3 µg/ml/24 h for both the Fab-scFv and the Fab-(scFv)2 molecules.

We conclude that C-terminal extension of the L and Fd chains with the various scFv does not hamper heterodimerization or secretion of the L:Fd heterodimer. Thus, the heterodimerization scaffold constitutes an instrument for efficient generation of disulfide-stabilized BsAbs and TsAbs of intermediate size (75 and 100 kDa, respectively) in mammalian cells.

Binding characteristics of Fab-scFv and Fab-(scFv)2 molecules

To verify the functionality of the individual binding moieties of the anti-tumor cell/anti-T cell Fab-scFv and Fab-(scFv)2 molecules, their binding characteristics on Ag-positive cells were determined by flow cytometry and compared with the binding characteristics of the mAbs from which the respective binding moieties were derived. As shown in Fig. 2Go, the (anti-hPLAP x anti-CD3) Fab-scFv molecule recognizes both hPLAP-transfected MO4I4 and CD3+ TE2 cells. Also the (anti-hPLAP x anti-CD3 x anti-BCL1) Fab-(scFv)2 derivative combines the binding characteristics of its donor mAbs and recognizes its Ags on MO4I4, TE2, and BCL1+ B cells. Thus both types of Ab derivatives retained the functionality and specificity of the constituting Fab and scFv components.



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  		FIGURE 2. Binding characteristics of Fab-scFv and Fab-(scFv)2 molecules reveal dual and triple Ag specificity. Binding of Fab-scFv, Fab-(scFv)2, and parental mAbs was assessed by indirect immunofluorescence and flow cytometry analysis (thick lines). Except for mAb 145-2C11, which was detected with FITC-conjugated anti-hamster Ig Ab, FITC-conjugated anti-mouse Fab was used for detection. Negative controls were stained with FITC-labeled secondary Ab alone (thin lines).

 
To verify whether the different binding moieties of the bispecific Fab-scFv and trispecific Fab-(scFv)2 are capable of concurrent binding to their respective Ags, we immobilized the BsAb and TsAb by binding to one of its Ags, the CD3 ligand on T cells, and detected this binding by the subsequent retention of the hPLAP Ag (Fig. 3GoA). In the TsAb, two more axes of bispecific binding can be determined, i.e., anti-hPLAP x anti-BCL1 and anti-CD3 x anti-BCL1. These concurrent bindings were assayed by detection of the TsAb bound to hPLAP+ MO4I4 cells or CD3+ TE2 cells with a biotinylated BCL1 Ag (which is a mouse IgM molecule) (Fig. 3GoB). All three approaches demonstrated multiple bispecific binding along the three different axes of the TsAb.



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  		FIGURE 3. Fab-(scFv)2 leads to bispecific binding along its three axes. A, Dual binding of the Fab-scFv BsAb (B) and the Fab-(scFv)2 TsAb (T) along the (anti-CD3 x anti-hPLAP) axis was demonstrated by detection of TE2 cell-bound BsAb or TsAb by cellular ELISA with hPLAP and a colorigenic hPLAP substrate. In negative controls (open columns), no Ab or cells were added or TsAb was replaced with the monospecific anti-hPLAP E6 mAb. B, Dual binding activity of the TsAb (anti-BCL1 x anti-hPLAP x anti-CD3) along the (anti-hPLAP x anti-BCL1) and (anti-CD3 x anti-BCL1) axes was demonstrated by indirect immunofluorescence. Binding of TsAb to hPLAP+ MO4I4 or CD3+ TE2 cells was detected using biotinylated BCL1 IgM mAb as secondary Ab and FITC-labeled avidin (filled curves). Negative controls were stained with secondary Ab and FITC-labeled avidin (open curves).

 
Fab-scFv fusion molecules mediate tumor cell-dependent activation of T cells

The capacity of the (anti-hPLAP x anti-CD3) BsAb and the (antiBCL1 x anti-hPLAP x anti-CD3) TsAb to recruit and activate CD3+ T cells by its anti-T cell and anti-tumor cell reactivity was examined on the basis of a tumor cell-dependent induction of T cell proliferation and T cell-mediated cytotoxicity. Due to its dual anti-tumor cell binding moieties, the TsAb was assayed in two experimental settings, one primed with the hPLAP+ MO4I4 tumor cells, and the other primed with the BCL1 tumor cells. As shown in Fig. 4Go, A and C, induction of both proliferative and cytotoxic T cell activities was apparent only in the presence of tumor cells, T cells, and BsAb or TsAb, but not in the absence of either of these components. Clearly, generation of T cell reactivity was dependent on tumor cell-induced cross-linking of the monovalent {alpha}-CD3 moiety of the BsAb or TsAb resulting from the interaction of the anti-tumor cell moiety with its corresponding tumor-associated Ag. The level of T cell reactivity depended on the amount of Ab added; the optimal concentration was determined to be between 0.1 and 1 µg/ml for both the BsAb and the TsAb (Fig. 4Go, B and D).



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  		FIGURE 4. Fab-scFv and Fab-(scFv)2 lead to cell-cell cross-linking and tumor cell-dependent activation of effector T cells. A, Mitomycin-treated MO4I4 or BCL1 cells were incubated with C3H or BALB/c syngeneic spleen cells, respectively, (E:T ratio 2:1) and 0.5 µg/ml BsAb (B) or TsAb (T) (filled columns). To prove specificity, selected components were omitted from the reaction (open columns). B, A titration curve starting from 1 µg/ml of the BsAb ({circ}) and the TsAb in a reaction with MO4I4 tumor cells ({diamondsuit}), or of the TsAb with BCL1 tumor cells ({blacktriangleup}) is shown. C, 51Cr-labeled MO4I4 cells or BCL1 cells were cocultured for 4 h with C3H or BALB/c syngeneic splenocytes, respectively, (E:T ratio 50:1) in the presence of BsAb (B) or TsAb (T) (filled columns). To prove specificity, selected components were omitted from the reaction (open columns). D, Titration curve starting from 1 µg/ml of BsAb (anti-hPLAP x anti-CD3) ({circ}) or TsAb assayed for its anti-hPLAP x anti-CD3 axis ({diamondsuit}) or its anti-BCL1 x anti-CD3 axis ({blacktriangleup}) of T cell cytotoxicity with either the labeled MO4I4 or BCL1 tumor cells as a target.

 
From these experiments we conclude that no conformational restrictions impair the concurrent cognitive interaction of the two binding moieties of the BsAb, and especially of the three binding moieties of the TsAb, to cross-link two different cells. As a consequence, these novel types of recombinant Abs exert activities typical of BsAbs, namely, recruitment and tumor cell-dependent activation of effector T cells. The TsAb can do this with two different Ags.

Stability of L:Fd heterodimerized bi- and tribodies

To be useful in therapy settings, recombinant Abs need to be sufficiently stable, and the recombinant model should not impair the functionality of the Ab. To check the TsAb for its possible tendency to form dimers or higher aggregates, we purified a His-tagged TsAb on a metal chelating column and subsequently applied the concentrated material on a size exclusion column (Fig. 5GoA). More than 95% eluted as a monomer at 100 kDa. A very small fraction of the TsAb was found as a dimer, but the material eluting in the void volume did not contain any TsAb (as determined by Western blotting and immunodetection, data not shown).



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  		FIGURE 5. Stability of Fab-scFv and Fab-(scFv)2 bi- and tribody. A, The TsAb (anti-BCL1 x anti-hPLAP x anti-CD3) was purified from cell culture supernatant and analyzed on a Coomassie Brillant Blue-stained SDS-PAGE and on a Superdex 200 XK 26/88 (Amersham Pharmacia Biotech). The column was calibrated with standard proteins of 669, 150, 43, 13, and 0.5 kDa. The material eluting in the void (Vo) mainly contained contaminating proteins and almost no TsAb. The peaks corresponding to monomeric (M) and dimeric (D) TsAb are indicated. B, Capture of the Tsab with target cells. i, Dilution series of the TsAb immunodetected after SDS-PAGE with anti-mouse IgG serum shows that 1% of the starting dose can still be detected. ii, The same amount of TsAb was washed three times with MO4I4 (hPLAP+), BCL1 (BCL1+), TE2 (CD3+), or SP2/0 (irrelevant) cells. iii, The same experiment was performed with the parental mAbs E6 (anti-hPLAP), B1 (anti-BCL1), and 145.2c11 (anti-CD3). C, In vitro stability of the bi- and tribody: 1 µg/ml of Ab fragment was incubated at 37°C for up to 26 h in either PBS or freshly prepared mouse serum. The stability was measured as the remaining activity in a T cell proliferation assay with MO4I4 (hPLAP+) cells, which accounts for the presence of two specificities (anti-hPLAP and anti-CD3).

 
To determine the fraction of purified material that is active, the TsAb was absorbed out by immunoprecipitation with target cells. The unabsorbed fraction was detected by Western blotting and quantified by comparison to a series of 2-fold dilutions of the unabsorbed sample (Fig. 5GoB). Capture of the TsAb by MO4I4 or BCL1 cells resulted in a 100-fold reduction of the amount of unbound TsAb. Similar results were obtained when the corresponding anti-hPLAP and anti-BCL1 mAb were immunoprecipitated. Compared with SP2/0 cells (used as a negative control), incubation with TE2 cells (CD3+) did not result in significant capture of the TsAb. However, the anti-CD3 mAb 145.2C11 was not captured effectively by the TE2 cells, likely as a consequence of the low CD3 expression levels on these T cells. In conclusion, this experiment shows that the large majority of the L-scFv:Fd-scFv heterodimers are present in a correct conformation.

We also determined the stability of the BsAb and the TsAb after incubation at 37°C in PBS or in freshly prepared mouse serum (Fig. 5GoC). The stability was measured as the remaining bispecific activity in the Ab samples that were incubated for different time intervals. Bispecific activity was assayed with a T cell proliferation assay. Regression analysis of the data obtained predicts half lives of the BsAb and TsAb of ~30 h when incubated in serum, as compared with 80 h when incubated in PBS (R2 > 0.9). These data indicate that the bibody and tribody format is compatible with therapeutic use in vivo.

Because the effect of two C-terminal fusions on the affinity of the Fab has not been documented yet, we compared the binding affinity of the Fab moiety in the TsAb with the original Fab prepared from the E6 mAb. According to the measured surface plasma resonance parameters listed in Table IGo, the affinity of the TsAb is very comparable to the affinity measured for the native Fab. This indicates that the C-terminal fusions do not induce conformational changes that effect the Ag-binding site. We conclude that the bi- and tribody molecules created have a low tendency to aggregate and can be considered as stable proteins, suitable for therapeutic use.


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  		Table I. Comparing affinities of natural Fab' and Fab-moiety in Fab-(scFv)2 fusion1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
For therapy, large BsAbs exhibit a better pharmacokinetic behavior as compared with Ab derivatives of low m.w. However, this advantage is compromised by the strongly reduced tissue penetration of large BsAbs, a feature in which small size Ab derivatives excel. Intermediate size BsAbs, larger than Fab and smaller than whole Abs, combine the high tissue penetration of small molecules with the slow whole-body clearance of large molecules, and hence may have a better format for therapeutic applications. The drawback of these recombinant BsAbs is their requirement for efficient dimerization, essential for optimal expression and ease of purification. In this respect, heterotypic CL-CH1 interaction was shown to be a promising scaffold for production of bispecific minibodies in E. coli (13).

We propose to improve the heterodimerization efficiency of the CL:CH1 domains by using a mammalian type of expression system. Mammalian cells are known to exert a more stringent quality control on proteins proceeding along the secretory pathway. In particular, they contain the endoplasmic chaperone BiP/GRP78 that mainly binds to the CH1 domain of the IgH (26) and much weaker to the L chain (27). In agreement with this strict control, we never observed secreted CH1 fragments or Fd chains by themselves in the culture fluids of transfected cells, whereas L chains alone were efficiently secreted. Normally, the interaction of L chains with IgHs displaces the associated BiP and thus frees the Abs for secretion. However, coexpression of CL with CH1 or Fd did not lead to secretion of a CL:CH1 or CL:Fd heterodimer. Similar, cotransfection of L chains, instead of CL, did not result in secretion of L:CH1 heterodimers. Enlargement of CH1 with VH, resulting in formation of a Fd chain, was necessary to obtain secretion of L:Fd heterodimers in cotransfected cells. These heterodimers represented over 90% of the secreted products, reflecting tight control of their secretion. These results indicate that interaction of CL with CH1 is not efficient to free the complex for secretion, and an additional VL:VH interaction is necessary to release the CH1 domain from its endoplasmic chaperone, thus leading to efficient secretion. A similar dependence on VL:VH interactions for BiP displacement was observed in a recent study of Lee and coworkers, showing that the BiP:Fd interaction could not be displaced when the VL or VH domains were mutated to prevent folding (28).

Having established coexpression of L and Fd as the minimal scaffold for efficient heterodimerization in mammalian cells, we analyzed the applicability of L:Fd heterodimerization for generating intermediate size BsAbs and TsAbs. To avoid possible sterical hindrance when cross-linking two cells, and to allow a better reach for distant Ags, the C terminus of the L and Fd chains was preferred over the N terminus to fuse scFv molecules. The resulting L:Fd-scFv bispecific heterodimer typically represented >90% of the secreted Ig pool. This represents a significant improvement over other expression systems, such as CL:CH1-driven heterodimerization of minibodies in E. coli, where 63% of the total Ig pool was bispecific (13). Furthermore, with the L:Fd template, efficient heterodimerization was achieved without introduction of antigenic heterodimerization domains or application of extensive postproductional processing. A final advantage, intrinsic to the present model, derives from the formation of a Fab binding moiety only in correctly folded heterodimers. Thus, BsAb or TsAb can easily be separated from irrelevant side products, such as L-scFv:L-scFv homodimers, by a single immunoaffinity purification step with the appropriate Ag. Also, an affinity purification directed against the heavy-chain fusion product would have the same result, because the Fd chain is always in the heterodimer format.

Both Fab-scFv and Fab-(scFv)2 molecules, which we refer to as bibody and tribody, were fully functional in bispecific binding and in cross-linking of effector T cells with tumor cells. Furthermore, tribodies exhibited dual binding along the three axes of the molecule, demonstrating reach and flexibility of the different binding sites. As a result, the same tribody induced T cell reactivity against two types of tumor cells by alternating use of its dual anti-tumor cell binding moieties (combined with its anti-CD3 moiety). The new bi- and tribody model was shown to have a low tendency to aggregate and to be stable in physiological conditions, making it suitable for therapeutic use.

Due to its high level of disulfide-stabilized and specific heterodimerization, this molecule is a useful alternative for generating BsAb and TsAb. The tribody model provides an easy way to construct monovalent TsAbs, which is most valuable considering the heterogeneity of most tumors. Another application can be found in increasing the avidity for the tumor antigen or for the tumor cell.

In summary, the highly efficient heterodimerization of L and Fd chains in mammalian cells constitutes an ideal platform for generating fully functional, disulfide-stabilized BsAbs and TsAbs of intermediate size by C-terminal enlargement with one or two scFv molecules. The dependence on mammalian expression renders these novel recombinant Ab derivatives suitable for production in mammalian cell factories or transgenic flock if needed as a therapeutic agent.


   Acknowledgments
 
We thank Dr. J. Demolder and Dr. W. Lammerant (Ghent University) for the use of unpublished plasmids. Dr. M. Hall (University of Birmingham, U.K.), Dr. M. De Broe (University of Antwerp, Belgium), and Dr. G. LeClercq (Ghent University) are acknowledged for donating HEK293T cells, MO4I4 cells, and 145.2C11 mAbs, respectively.


   Footnotes
 
1 This work was supported by the Inter-University Network for Fundamental Research (Belgium) Program. R.S. was recipient of a fellowship from Vlaams Instituut voor de Bevordering van Het Wetenshappelijk Technologisch Onderzoek in de Industie (IWT) and Vlaamse Liga tegen Kanker. A.W. is a research assistant with the Fonds Wetenschappelijk Onderzoek-Vlaanderen and S.S. with IWT. Back

2 Address correspondence and reprint requests to Dr. J. Grooten, Department of Molecular Biology, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Back

3 Abbreviations used in this paper: BsAb, bispecific Ab; hPLAP, human placental alkaline phosphatase; scFv, single-chain variable fragment; TsAb, trispecific Ab; BCL, B cell lymphoma. Back

Received for publication October 15, 1999. Accepted for publication September 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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