Highly Enhanced Cytotoxicity of a Dimeric Bispecific Diabody, the hEx3 Tetrabody*

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.

Bispecific antibodies (BsAbs) 2 are recombinant antibodies that can bind to two different antigenic epitopes. Bispecificity can be used in cancer immunotherapy to cross-link tumor cells to immune cells such as cytotoxic T cells, natural killer cells, and macrophages. This cross-linking accelerates the destruction of the tumor cells by the immune cells, which may translate into improved antitumor therapy and lower production costs by decreasing the doses needed (1,2). However, the use of BsAbs in clinical studies has been hampered by difficulties in producing them on a large scale. Conventional chemical conjugation has been used, but the quality of the antibody produced is inconsistent (3). The production of BsAbs by somatic fusion of two hybridomas to form a quadroma yields BsAbs of more consistent quality but results in the formation of various chainshuffled antibodies; for instance, 10 different antibodies can be generated after random association of two heavy and two light chains (4,5).
Advances in recombinant technology have made it feasible to generate small recombinant BsAbs constructed from two different variable antibody fragments. Bispecific diabodies are the smallest available BsAbs, and the distance between the two antigen-binding sites is sufficient to link two cells (6,7). The effectiveness of bispecific diabodies in cancer therapy has been shown extensively in in vitro and in vivo models (8 -10). We also have constructed functional bispecific diabodies (11,12). In particular, the humanized bispecific diabody hEx3 has marked antitumor activity and can retarget lymphokine-activated killer cells with the T cell phenotype (T-LAK cells) against epidermal growth factor receptor (EGFR)-positive cell lines (13,14). The compact structure of bispecific diabodies contributes to low immunogenicity, high tumor penetration, and the potential for large scale preparation through bacterial expression systems; however, the downsizing results in rapid clearance from blood. In addition, the structure contains only one binding domain for each target, which results in low functional affinity (15,16).
Multimerization of small recombinant antibodies is one available strategy for improving their pharmacokinetic and binding affinity. In single-chain Fvs (scFvs), the length and composition of the polypeptide linker between the variable heavy (VH) and light (VL) domains strongly influence the formation of the multimeric structure. A linker of 15 amino acid residues leads to the formation of an scFv, but reducing the linker length to 8 -12 residues causes the scFvs to assemble into dimers, so that diabodies are formed. A further reduction to less than five residues leads to the formation of scFv trimers or tetramers (known as triabodies or tetrabodies) (17)(18)(19)(20)(21). These scFv multimers are larger and have higher valency than the monomeric form; consequently, their clearance from circulation and accumulation on tumors are improved (22,23).
Bispecific diabodies are generally produced from heterodimerization of two different hetero-scFvs (e.g. VH A -VL B and VH B -VL A ) with a glycine-rich linker (GGGGS; 8,24). The hetero scFvs can also form higher multimeric structures (25), and the multimeric bispecific diabodies formed are expected to have multivalent bispecificity and appropriate molecular weight. Here, we examined the multimerization of hEx3 by preparing monodisperse tetramers (hEx3 tetrabodies). These bispecific tetrabodies had much higher affinity for each antigen than normal diabodies due to an avidity effect, which led to strong inhibition of cancer cell growth. To our knowledge, this is the first detailed quantitative characterization of functional bispecific tetrabodies.

EXPERIMENTAL PROCEDURES
Preparation of Recombinant BsAbs-For the expression and preparation of hEx3, we used three different methods in accordance with previous reports: a preparation using a bacterial expres- The elution volume is noted on the x axis. A, hEx3 from a refolding system (13); B, hEx3 from a mammalian expression system (14); and C, hEx3 from an Fc fusion format (27). D, SDS-PAGE analysis of the eluted fractions under reducing conditions. The tetramer (T) and dimer (D) fractions of hEx3 from the Fc fusion format are shown. mAU, milli-absorbance unit.   sion and in vitro refolding system (13), a preparation using a mammalian expression system (14), and a preparation using Fc fusion format and restriction protease digestion (26,27). Size-exclusion chromatography with a HiLoad Superdex 200-pg column (26/60; GE Healthcare) was used to fractionate each prepared hEx3 solution. The column was equilibrated with phosphate-buffered saline (PBS), and then 5 ml of purified recombinant antibodies was applied to the column at a flow rate of 2.5 ml/min. Dynamic Light Scattering and Static Light Scattering Measurements-Dynamic light scattering (DLS) and static light scattering (SLS) measurements were carried out at 20°C on a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK) using a He-Ne laser ( ϭ 633 nm). All of the antibody solutions were filtered through a polytetrafluoroethylene filter. For DLS, the antibody solutions at 15 M were measured using a noninvasive back-scatter optical system, and the correlation curve was fitted using the default exponential g2() fit function to estimate the hydrodynamic diameters of the antibodies. For analyzing molecular weight, SLS of the antibody solutions at 0.3-1.0 mg/ml was measured, and a Debye plot was made using the scattering intensity.
In Vitro Growth Inhibition Assay-T-LAK cells were induced as reported previously (28). In brief, peripheral blood mononuclear cells were cultured for 48 h at a density of 1 ϫ 10 6 cells/ml in a medium supplemented with 100 international units/ml of recombinant human interleukin-2 (kindly supplied by Shionogi Pharmaceutical Co., Osaka, Japan) in a culture flask (A/S Nunc, Roskilde, Denmark) that was precoated with anti-CD3 monoclonal antibody (10 g/ml).
Isothermal Titration Calorimetry (ITC)-Thermodynamic analyses for the interactions of recombinant  antibodies for soluble EGFR (sEGFR) and CD3 were performed by microtitration calorimetry using a VP-ITC from MicroCal Inc. (Northampton, MA) (29). The method for expression and purification of sEGFR has been described previously (30). The expression vector for CD3 was kindly provided by Dr. Katsumi Maenaka (Kyushu University), and a preparation of CD3 was performed according to the previous report (31). Stability Tests-To examine in vitro stability, hEx3s were preincubated at 37°C for 1 h in human plasma. Growth inhibition relative to untreated hEx3s was then evaluated with the MTS assay.
Gel filtration analysis with a HiLoad Superdex 200-pg column (10/300) was used to evaluate the long term stability of the hEx3 tetramer in storage. After storage for 1 month at 4°C, 250 l of fractionated hEx3 tetramers was applied to a column equilibrated with PBS at a flow rate of 0.5 ml/min.

RESULTS
Structural Analysis of Prepared hEx3-We prepared the small recombinant bispecific antibody hEx3 using three different methods: refolding from insoluble aggregates expressed in Escherichia coli, secretory expression by Chinese hamster ovary cells, and Fc fusion expression by Chinese hamster ovary cells. Size-exclusion chromatography of each hEx3 preparation showed the predominant formation of dimers, but multimeric forms were also observed. The proportion of multimers varied with the method of preparation; refolded hEx3 produced only a small amount of multimers (Fig. 1A), whereas the secretory preparation using Chinese hamster ovary cells promoted the formation of multimeric forms, which corresponded to the fraction position of tetramers (Fig. 1B). Thus, hEx3 predominantly formed dimers but has the potential to form tetramers. hEx3 prepared from the Fc fusion format via restriction protease digestion also formed tetramers (Fig. 1C), and this method enabled the preparation of sufficient amounts of dimers and tetramers for further evaluation. The final yields of dimers and tetramers are 5 mg and 1 mg/liter culture, respectively. An SDS-PAGE analysis of the fractionated hEx3 showed that both the anti-EGFR VH-linker-anti-CD3 VL (h5HhOL) and anti-CD3 VH-linker-anti-EGFR VL (hOHh5L) hetero-scFv fragments formed equal proportions of dimers and tetramers with all the expression methods (SDS-PAGE for hEx3 from the Fc fusion is shown in Fig. 1D as a representative example).
To confirm the formation of tetramers, we employed DLS and SLS spectroscopy to quantify the size and molecular weights of the dimers and tetramers fractionated from hEx3 prepared with the Fc fusion format. An IgG-type mouse anti-CD3 antibody, OKT3, was used as a control for comparison. Both the dimer and tetramer had narrow distributions, centered at 3.4 and 6.6 nm, respectively; the size of tetramer was about twice that of dimer and two-thirds that of IgG ( Fig. 2 and Table 1). SLS measurement supported the molecular weight estimated from size-exclusion chromatography. Therefore, the multimer at the 190-ml fraction formed a monodisperse tetramer with equal amounts of h5HhOL and hOHh5L scFvs.
Growth Inhibition Effect of Each Fraction in hEx3 Solution-To analyze the influence of the tetramerization on the inhibition of human carcinoma cell growth, we analyzed prepared dimeric and tetrameric hEx3s with MTS. In the presence of T-LAK cells, both hEx3 forms strongly inhibited the growth of TFK-1 cells, but the tetramer was effective at a much lower  JULY 2, 2010 • VOLUME 285 • NUMBER 27 concentration, 10 fmol/ml (Fig. 3A). When peripheral blood mononuclear cells were applied as effector cells, although high concentrations of hEx3s were required, the tetramer also inhibited more effectively than the dimer (Fig. 3B). Thus, the multimerization into tetramers increased the function of hEx3.

Characterization of a Bispecific EGFR ؋ CD3 Tetrabody
Thermodynamic Analysis of Each Fraction of hEx3-To investigate the binding stoichiometry of each fraction in hEx3 for EGFR and CD3, we performed thermodynamic analyses by ITC. IgG and Fab were used as control molecules with bivalent and monovalent binding, respectively. The binding constants and stoichiometry are summarized in Table 2. Dimeric hEx3 showed 1:1 stoichiometric binding for EGFR (Fig. 4C) and CD3 (Fig. 4D), similar to Fab, indicating that the anti-EGFR Fv and anti-CD3 Fv portions in the dimeric hEx3 had formed correctly; that is, the prepared dimers were monomorphous diabodies without inactive homodimers. This result is consistent with our previous results supporting the formation of monomorphous hEx3 diabodies (14). In the case of tetramers, the integration plots for EGFR and CD3 showed the same binding stoichiometry as IgG but not Fab, that is, a 1:2 stoichiometric binding for both antigens (Fig. 4, C  and D). The tetramer therefore formed monomorphous hEx3 tetrabodies with bivalency for two individual targets.
Comparison of Binding Kinetics with Surface Plasmon Resonance-To confirm the effect of the multivalency of tetrameric hEx3, we evaluated the binding kinetics for immobi-lized sEGFR by surface plasmon resonance. The binding kinetics for CD3 were not determined, because the CD3 receptors were inactivated when immobilized on a sensor chip. The sensorgram for tetrameric hEx3 against EGFR showed an association curve similar to that of the dimer, but the dissociation of the tetramer was slower than that of the dimer (Fig. 5). A global fitting to a 1:1 interactional model with a mass transport term indicated that the tetramers had an association rate similar to that of the dimers, but the dissociation rate was one-seventh of the dimer rate (Table 3); consequently, the affinity constant of the tetramer was 17-fold that of the dimer. The multimerization into a tetramer influenced the dissociation process of diabodies, resulting in increased affinity for the antigen.
Stability Test under Physiological Conditions-Physiologic stability is a critical factor for potential therapeutic recombinant proteins. Therefore, we examined the cytotoxicity of dimeric and tetrameric hEx3 after preincubation at 37°C in human plasma. The activity of the dimers was slightly reduced; however, the intense cytotoxicity of the tetramer was retained (Fig. 6A). The stability of assembled structure was also evaluated by size-exclusion chromatography of the tetramer after storage for a month (Fig. 6B). Although a few tetramers were converted into dimers, we confirmed that the tetrameric structure was sufficiently stable for use after 1 month in storage.

DISCUSSION
Bispecific diabodies show several advantages over BsAbs produced from hybrid hybridomas or chemical conjugation; however, bispecific diabodies are also cleared rapidly, and their decrease in valence generally causes low functional affinity (15,16). Shortening the middle linker in scFv leads to self-multimerization, and the multimerization can improve the pharmacokinetics and increase functional affinity due to an avidity effect (17)(18)(19)(20). Multimerization of bispecific diabodies has been observed previously (25), but the effectiveness of bispecific diabodies has not been studied to date.
In the present study, we found that hEx3 formed multimers. We purified these multimers, and kinetic and thermodynamic analyses of each hEx3 fraction quantitatively demonstrated that the tetramer had two functional binding sites for each antigen ( Fig. 4 and Table 2). This increase in binding sites provided strong growth inhibition activity. The multimerization was effective even for bispecific diabodies.
Engineering of linkers in singlechain diabodies (scDbs), in which two hetero-scFvs are tandemly conjugated, can provide tetravalent bispecific dimers called tandem scDbs (tanDbs). The tanDbs exhibit not only higher functional affinity  and stability under physiological conditions in vitro than scDbs but also longer blood retention and higher therapeutic effects in vivo (15,32,33). In this study, we prepared highly functional bispecific tetrabodies from hetero-scFv fragments with molecular sizes approximately half those of scDbs. Although the bispecific tetrabodies formed as a by-product of bispecific diabodies, the formation from smaller fragments might be an advantage in protein expression. In contrast to the structure of tanDbs, in which all four variable domains of one chain interact with the variable domains of the second chain (33,34), the structure of the hEx3 tetramer is probably a circular structure, similar to that of the scFv tetramer (known as a tetrabody; Refs. 18,35). We previously reported a strong interdomain interaction between the cognate VH and VL domains of hEx3 (14); this strong interaction probably contributes to the formation of a stable circular structure for the hEx3 tetrabody with four active binding sites. To date, several different small BsAb formats have been proposed to increase efficacy and availability, including not only scDb and tanDb but also tandem scFv (36) and minibodies (37). Bispecific tetrabodies like the hEx3 tetramer also should be considered small BsAb formats for the development of effective cancer therapeutic antibodies. Although in vivo experiments with the hEx3 tetramer are now under way, we did confirm their stability in physiologic conditions and in long term storage (Fig. 6).
In conclusion, we showed that the multimeric molecules in hEx3 solution were homogenous tetramers with high cytotoxicity. The multimerization of small antibody fragments can lead to improved pharmacokinetics and binding affinity, resulting in an enhancement of the therapeutic effect. To increase the population of hEx3 tetramers for therapeutic application, we are working to modify the middle linker in hetero-scFvs, to change the orientation of VH and VL, and to create mutations to minimize the steric interference in the tetrameric form, similar to how the scFv multimer has been modified (35,38,39).