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The Journal of Immunology, 2001, 166: 6112-6117.
Copyright © 2001 by The American Association of Immunologists

Increasing the Affinity for Tumor Antigen Enhances Bispecific Antibody Cytotoxicity1

Adrian M. McCall2,3,*, Lillian Shahied3,*, Anne R. Amoroso*, Eva M. Horak*, Heidi H. Simmons*, Ulrick Nielson{dagger}, Gregory P. Adams*, Robert Schier4,{dagger}, James D. Marks{dagger} and Louis M. Weiner5,*

* Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111; and {dagger} Department of Anesthesia, University of California, San Francisco, CA 94110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10-7 to 10-11 M, were fused to the phage display-derived NM3E2 human scFv. NM3E2 associates with the extracellular domain of human Fc{gamma}RIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of 51Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu KD = 1.7 x 10-7 M, 14.5% with KD = 5.7 x 10-9 M, and 21.3% with KD = 1.7 x 10-10 M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG Bsab. Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Fc domains of many IgG Abs interact with human leukocyte Fc receptors to stimulate Fc receptor-dependent leukocyte activation (1). In the process of Ab-dependent cellular cytotoxicity (ADCC),6 Abs bridge Fc receptor-bearing leukocytes and Ag-bearing target cells, resulting in cytotoxicity directed against the targets. This process has been observed with numerous mAbs directed against malignant cells and has formed the basis for human clinical trials of anti-tumor mAb used alone or in combination with cytokines that potentiate ADCC (2, 3, 4, 5). However, the majority of these trials have failed to yield high clinical response rates, even when Abs that efficiently mediate ADCC have been used (6, 7). To improve Ab-targeted antitumor cytotoxicity, bispecific Abs (Bsab) have been developed that specifically target both tumor Ags and activating epitopes on cytotoxic leukocytes such as Fc{gamma}RI (CD64) (8), Fc{gamma}RIII (CD16) (9, 10), CD44 (11), CD3, the {alpha}{beta} TCR complex (12, 13), and CD28 (14). Since Bsab can target triggering epitopes that are distinct from sites on the Fc{gamma} receptor that engage IgG Fc{gamma} domains, such molecules are not limited by competition from circulating host Abs for binding to activating epitopes. Bsab have been constructed as chemically linked heterodimers (15) by purification from hybrid hybridoma supernatants (16) and more recently as recombinant fusion proteins (17, 18, 19, 20, 21). Many Bsab exhibit potent in vitro cytotoxicity properties and promote tumor lysis far more efficiently than do the conventionally structured IgG molecules from which they were derived, even in the presence of competing host Igs (10). These attributes have led to the clinical evaluation of several Bsab that target tumor Ags and either Fc{gamma}RI (22), Fc{gamma}RIII (10), or CD3 (23, 24). Modest clinical activity has been seen in these studies, and several avenues for modification of Bsab structures and treatment strategies have been identified (22, 25, 26, 27).

We have extensively studied the 2B1 Bsab in preclinical studies and in a series of human clinical trials. 2B1 was prepared by the fusion of the 520C9 and 3G8 hybridomas to yield a highly purified IgG1 murine Bsab recognizing epitopes on the extracellular domains of the HER2/neu tumor Ag and human Fc{gamma}RIII (CD16), respectively. This Bsab efficiently mediates in vitro cytotoxicity against HER2/neu-expressing human tumor cells by human NK cells and macrophages (11, 10) and possesses potent antitumor activity in a human tumor xenograft model in immunodeficient mice (28). In a phase I clinical trial of 2B1, the maximally tolerated dose was surprisingly low (2.5 mg/m2), largely due to thrombocytopenia, fevers, hypotension and other manifestations of the "first-dose effect" frequently observed with intact IgG Ab therapy (25). These toxicities have been shown to result at least partially from the simultaneous engagement of human Fc{gamma}RIII and Fc{gamma}RII on leukocytes by the Bsab via its anti-Fc{gamma}RIII and Fc domains, Ñleading to receptor cross-linking and systemic leukocyte activation (29). We postulated that the elimination of Fc domains would reduce this process and permit the use of higher Bsab doses, improving tumor delivery and therapeutic outcome. This was accomplished by isolating single-chain Fv fragments (scFv) from a human phage display library reactive with HER2/neu and Fc{gamma}RIII, respectively (30, 21) and fusing these proteins using an amino acid linker to create a recombinant (scFv)2 Bsab (21). In this study, we show that this Bsab and its HER2/neu affinity mutants do not induce leukocyte activation in the absence of engagement of HER2/neu-expressing tumor cells. We also postulated that increased affinity for HER2/neu would be associated with more efficient Bsab promotion of cytotoxicity. This was tested using Bsab containing the same anti-Fc{gamma}RIII scFv but using anti-HER2/neu scFv targeting an identical epitope with affinities ranging from 10-7 to 10-10 M. Bsab with increased affinity for HER2/neu promoted more targeted tumor cytotoxicity than their lower affinity variants. These findings provide a platform for the construction of Bsab with improved selective cytotoxicity properties.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of anti-human (scFv)2 Bsab recognizing the extracellular domains of HER2/neu and Fc{gamma}RIII

We have previously reported on the construction of a recombinant human (scFv)2 Bsab recognizing the extracellular domains of HER2/neu and human Fc{gamma}RIII (CD16). Both scFvs were isolated from human phage display libraries (21). This Bsab, designated C6.5-NM3E2, binds to HER2/neu with an affinity of 6.8 x 10-9 M and to human Fc{gamma}RIII ECD with an affinity of 2.4 x 10-7 M by surface plasmon resonance. Four additional bispecific scFvs were produced, using affinity mutants of C6.5 that also have been described previously. The anti-HER2/neu scFvs used were C6G98A (KD = 3.1 x 10-7 M), C6ML3.9 (KD = 1.0 x 10-9 M), C6MH3B1 (KD = 1.2 x 10-10 M), and C6B1D2 (KD = 1.5 x 10-11 M). The generation of the Bsab molecules was performed using the pHENIX vector that contains two cloning sites separated by a Ser2(Gly4Ser)2Gly2Ser linker sequence (31). To create each Bsab, the C6.5 variant scFv genes were PCR amplified and cloned into the first position of the pHENIX vector using primers with the restriction sites NcoI and XhoI. The NM3E2 scFv was PCR amplified and cloned into the second position using primers containing the restriction sites ApaLI and NotI. The NcoI/NotI fragments containing the C6.5 variant-NM3E2 sequences were then excised and cloned into the vector pUC119 myc his, as described elsewhere (30).

The Bsab were expressed as described previously (21). Briefly, TG1 Escherichia coli were transformed with the pUC119 myc his plasmid containing the Bsab DNA to express the Bsab protein. Transformed cells were inoculated into 20 ml 2x YT/100 µg/ml ampicillin (Sigma, St. Louis, MO )/1% (w/v) glucose and incubated overnight at 37°C in a shaking incubator at 250 rpm. The overnight culture was used to inoculate 2 liters of 2x YT/100 µg/ml ampicillin/0.1% (w/v) glucose and was grown at 37°C until it achieved an A600 ~1.0. Isopropyl {beta}-D-thiogalactopyranoside (Fisher Scientific, Pittsburgh, PA) was added to a final concentration of 1 mM to induce expression of the scFvs. The culture was transferred to a 30°C incubator for 4 h and then harvested by centrifugation at 4000 x g for 20 min. Fifty milliliters of ice-cold periplasmic extraction buffer (30 mM Tris-HCl (pH 8.0), 20% (w/v) sucrose, 1 mM EDTA) was added to the bacterial pellets for resuspension. The bacteria were centrifuged again at 4000 x g for 20 min, and the supernatant was retained. Fifty milliliters of ice-cold osmotic shock buffer (5 mM MgSO4) was added to the bacterial pellets. The preparation was again centrifuged at 4000 x g for 20 min and the supernatant was retained. Supernatants from the periplasmic and osmotic shock extractions were pooled and centrifuged at 17,500 x g for 20 min to remove cellular debris. The resulting supernatants were dialyzed overnight against PBS at 4°C.

The Bsab were purified by immobilized metal affinity chromatography and HPLC size exclusion chromatography as described previously (21). One milliliter of Ni-NTA-agarose (Qiagen, Dusseldorf, Germany) was added to the dialyzed supernatant. The solution was mixed for 1 h at 4°C and then centrifuged at 1750 x g for 20 min. After aspirating all but 50 ml of the supernatant, the remaining fluid was removed by centrifugation at 1750 x g for 10 min in a 50-ml polypropylene centrifuge tube (Corning, Corning, NY). The pellet was washed three times with 10 ml of wash buffer (50 mM phosphate buffer (pH 7.5), 500 mM NaCl, and 20 mM imidazole at 4°C). Between each wash, the Ni-NTA-agarose was pelleted by centrifugation at 1750 x g for 5 min. The Bsab were eluted from the Ni-NTA-agarose with 2 ml of ice-cold elution buffer (50 mM phosphate buffer (pH 7.5), 500 mM NaCl, and 250 mM imidazole). The 2-ml elution volume was reduced to 500 µl by centrifugation in a Centricon 10 concentrator (Amicon, Beverly, MA). The Bsab were further purified by size exclusion chromatography on a Dynamax HPLC System (Rainin Instruments, Emeryville, CA) equipped with a Superdex 75 column (Pharmacia Biotech, Uppsala, Sweden). Fractions containing the Bsab were collected following gel filtration.

Affinity determination

The kinetics of the binding of C6.5 variant-NM3E2 (scFv)2 Bsab to the extracellular domain (ECD) of Fc{gamma}RIII and HER2/neu were determined by surface plasmon resonance using the BIAcore 1000 biosensor system (Biosensor, Uppsala, Sweden) (32). Human Fc{gamma}RIII and HER2/neu ECDs in 10 mM sodium acetate (pH 5.2) were immobilized on research grade CM5 sensor chips (Biosensor) by amine coupling (kit supplied by the manufacturer). Unreacted moieties on the chip surfaces were blocked with ethanolamine. Human Fc{gamma}RIII ECD (20 µg/ml) and human HER2/neu ECD (25 µg/ml) were applied to CM5 sensor chips at flow rates of 10 µl/min for 4 min, resulting in the immobilization of 343 response units on the Fc{gamma}RIII chip and 491 response units on the HER2/neu chip. The C6.5 variant-NM3E2 Bsab were dialyzed in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% (v/v) Surfactant P-20 (Biosensor) and then diluted into concentrations ranging from 0.98 to 1800 nM. Binding affinities were analyzed by continuous flow across each flow cell at a rate of 15 µl/min. Each sample was also passed over a control flow cell, which had been activated but contained no Ag. The control binding curves were subtracted from the corresponding test curves. Following the analysis of each sample, the CM5 sensor chip was regenerated with 4 M MgCl2, followed by 50 mM triethylamine at a flow rate of 50 µl/min. Kinetic analysis was performed for each molecule to determine the association (kon) and dissociation (koff) rates as well as the equilibrium constants (KA and KD).

Cytotoxicity assays

These studies were performed as previously described (10). Briefly, PBL were isolated by Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation and adherence of monocytes to gelatin/plasma-coated T-75 flasks. PBL were washed twice with PBS and once with RPMI 1640 and were found to be >95% viable using trypan blue dye exclusion. The PBL were treated with IL-2 (a gift from the Chiron Therapeutics, Emeryville, CA) at a concentration of 1000 U/ml for 3 h in supplemented RPMI 1640 (RPMI 1640, 6 g/L HEPES, 2 g/L NaHCO3, 10% (v/v) heat-inactivated FBS, 5 U/ml penicillin, 5 µg/ml streptomycin (Life Technologies, Rockville, MD), 10 µg/ml gentamicin, 0.29 mg/ml glutamine (Life Technologies), and 0.2 U/ml insulin (Novo-Nordisk, Clayton, NC)). SKOV-3 target cells (2 x 106) were labeled with 200 µCi of Na51CrO4 (NEN, Life Science Products, Boston, MA) for 1 h at 37°C in supplemented RPMI 1640. The 51Cr-labeled SKOV3 target cells were washed twice and 104 cells were added to individual wells of 96-well flat-bottom plates (Costar, Cambridge, MA) containing PBL and/or Bsab in supplemented RPMI 1640. Effector cells were added to yield E:T ratios ranging from 50:1 to 1:1 in the presence or absence of various concentrations of Bsab. Each well contained a total volume of 200 µl and all assays were performed in triplicate. The plates were centrifuged at 300 x g for 3 min, incubated for 4 h in a 5% (v/v) CO2 incubator at 37°C, and then centrifuged again at 300 x g for 3 min. One hundred microliters of supernatant was removed from each well for counting on a gamma 4000 counter (Beckman Instruments, Columbia, MD). Cytotoxicity was estimated by measuring the quantity of label released into culture supernatants using the formula: percent lysis = (experimental release (cpm) - spontaneous release (cpm))/(total added counts (cpm)/2 - spontaneous release (cpm)) x 100, where the experimental release was defined as cpm released by target cells in wells in the presence of either effector cells and/or Bsab, and the spontaneous release was defined as cpm released by target cells alone. The relative potency reported in Table IIIGo represents the calculated area under the log10 curves (percent lysis - concentration) over a wide concentration range of cytotoxicity potentiation in comparison to 2B1. The 2B1 equivalent lysis is defined as the Bsab concentration required to achieve lysis equivalent to that seen when 10 pM 2B1 is used.


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  		Table III. Effects of affinity on Bsab cytotoxicity efficiency

 
Calcium flux assay

The propensity of the C6BID2-NM3E2 (scFv)2 Bsab to trigger cellular activation in the absence of tumor cell engagement was examined in calcium flux flow cytometry-based assays, as described elsewhere (33). Polymorphonuclear neutrophils (PMN) and PBMC were isolated using Polymorphprep (Life Technologies, Gaithersburg, MD) density centri-fugation medium in a procedure similar to that described above. Contaminating RBC were lysed with hypotonic 0.2% NaCl and the remaining cells were equilibrated with an equal volume of 1.6% NaCl. Washed PMN and PBL were pooled and loaded with 4 µM fluo-3-acetylmethyl ester (fluo-3-AM; Molecular Probes, Eugene, OR) and 10 µM Fura Red AM ester (Molecular Probes) and DMSO (Sigma) in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS. The cells were incubated at a concentration of 1 x 107/ml for 45 min at 37°C and washed once with supplemented RPMI 1640. Just before acquisition, the cells were washed once and resuspended in PBS/3% (v/v) heat-inactivated FBS. FACS analysis was performed on a Becton Dickinson FACScan (BD Biosciences, Mountain View, CA) as described previously (10). Cells were collected at baseline (0 s). The mAb 3G8 (anti-human Fc{gamma}RIII), the Bsab 2B1 (murine IgG1, anti-human HER2/neu x anti-human Fc{gamma}RIII; Chiron Therapeutics), C6B1D2-NM3E2 (scFv)2 Bsab, or MOPC-21 (murine IgG1, isotype control) were added at final concentrations of 10 µg/ml, and cells were collected at 10-s intervals thereafter for up to 5 min. Ten to 20,000 cells were collected per sample. Analysis gates were set on PMN and PBL populations using forward and right angle light scatter. Both dyes are excited at 488 nm and mean fluorescence intensity (MFI) values were measured on a log scale. The fluorescein-based fluo-3 dye fluoresces with increasing intensity in the green region when bound to free Ca2+, whereas the Fura Red dye exhibits inverse behavior, with decreasing fluorescence intensity in the red region when bound to Ca2+. fluo-3 MFI/Fura Red MFI ratios were calculated for each cell population to directly determine relative intracellular Ca2+ mobilization (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein characterization and binding kinetics

The bispecific scFv were produced with yields ranging from 0.14 to 0.70 mg/L following purification and exhibited no significant aggregation and negligible dimerization. All constructs were expressed and purified in an identical manner and showed similar migration properties by SDS-PAGE as well as gel filtration (data not shown). Surface plasmon resonance was performed to determine whether the Bsab format had affected the binding of the individual scFvs to their respective Ags and to verify that the succession of affinities observed among the anti-HER2/neu scFvs remained unchanged. Association (kon) and dissociation (koff) rates as well as equilibrium constants (KA and KD) were determined for each molecule. Results showed that although the binding affinity trends observed with the anti-HER2/neu scFvs were maintained in the Bsab format, a reduction in overall affinity was observed for each mutant (see Table IGo). The association rates for binding to HER2/neu ECD were generally slower for all of the Bsabs with the exception of the C6.5B1D2-NM3E2 molecule, which associated at approximately the same rate as the C6.5B1D2 scFv (kon = 5.8 x 105 M-1 s-1 vs 4.8 x 105 M-1 s-1; see Table IGo). However, the rates of dissociation for the C6.5B1D2-NM3E2 and the C6.5G98A-NM3E2 molecules from HER2/neu ECD were faster than their corresponding monomeric scFvs (koff = 9.8 x 10-5 s-1 and 2.2 x 10-2 s-1). The C6.5-NM3E2, C6ML3.9-NM3E2, and C6.5B1-NM3E2 mutants dissociated at nearly the same rates as the parental scFv molecules (koff = 2.4 x 10-3 s-1, 4.0 x 10-4 s-1, and 2.0 x 10-4 s-1 respectively; see Table IGo).


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  		Table I. Binding to HER2/neu ECD; plasmon resonance-based affinity determinations of C6 series affinity mutants

 
A decrease in overall binding affinity was also observed in the interaction between the NM3E2 scFv segment of each Bsab and the Fc{gamma}RIII ECD (see Table IIGo). The KD values ranged from 1.0 x 10-7 M for C6BID2-NM3E2 to 6.0 x 10-7 M for C6.5B1-NM3E2. These values are ~10-fold less than the NM3E2 scFv affinity for the Fc{gamma}RIII ECD (2.0 x 10-8 M).


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  		Table II. Binding to Fc{gamma}RIII ECD: plasmon resonance-based affinity determinations of NM3E2 and NM3E2-based Bsab

 
Cytotoxicity

Each of the Bsab was evaluated for its ability to induce tumor lysis using a standard 4-h 51Cr release assay. In this assay, HER2/neu-positive 51Cr-labeled SKOV3 cells were incubated with IL-2-treated PBL with and without Bsab at E:T ratios ranging from 50:1 to 1:1. 2B1, an intact bispecific IgG that induces tumor lysis, served as a positive control. At a 50:1 E:T ratio, the ability of each Bsab to induce tumor lysis correlated with its affinity for the HER2/neu Ag (Fig. 1Go). For example, C6B1D2-NM3E2, which possesses the highest affinity for HER2/neu (KD = 1.7 x10 –10 M), induced a maximal level of 21% tumor lysis at an 18 nM Ab concentration. However, the Bsab with the lowest affinity for HER2/neu, C6G98A-NM3E2 (KD = 1.7 x 10-7 M), maximally stimulated lysis of 6.5% at a 180 nM concentration. The mutants possessing moderate affinity for HER2/neu (C6.5-NM3E2, C6 ML3.9-NM3E2, and C6B1-NM3E2) also promoted peak tumor cytotoxicity at a 180 nM concentration, but with 15.3, 14.9, and 18.1% lysis, respectively (C6.5-NM3E2 and C6B1-NM3E2 not shown). Shown in Table IIIGo are the potencies for each Bsab relative to 2B1 as well as the 2B1-equivalent lysis concentrations. These data were derived from several experiments (n >= 3 for each Bsab) with each Bsab compared with 2B1. As expected, a higher concentration of the lower affinity molecules was required to obtain a cytotoxic effect equivalent to 2B1. Fig. 2Go shows the percent lysis promoted by each Bsab (at a representative concentration of 18.2 nM) at three different E:T ratios. Unlike the 50:1 ratio, where a distinct lysis gradient correlated with HER2/neu affinity, the Bsab exhibited less variation in the induction of cytotoxicity at 25:1 and 5:1 ratios.



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  		FIGURE 1. ADCC profiles of Bsab at different molar concentrations. A constant E:T ratio of 50:1 is displayed. This experiment is representative of two such experiments performed with all of the Bsab at once and was confirmed 3–10 times in experiments conducted using each of the (scFv)2 Bsab molecules alone in comparison to 2B1. All SEM were within 15% of the mean percent lysis values, except for 0.181 nM C6ML3.9-NM3E2 (SEM, 28%).

 


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  		FIGURE 2. Cytotoxicity profiles of Bsab at different E:T ratios at a concentration of 18.2 nM of each Bsab. Each point represents the mean ± SEM for the three values.

 
Intracellular Ca2+ mobilization assay

Flow cytometry-based intracellular Ca2+ mobilization assays were performed to determine whether the Bsab activated Ca2+-sensitive, dye-loaded PMN in the presence of PBMC. The fluo-3/Fura Red ratios for the PMN indicated that addition of the C6B1D2-NM3E2 Bsab did not trigger Ca2+ mobilization (Fig. 3Go), as there was a steady decrease in the ratio from baseline over the 5-min period, similar to findings with the negative control MOPC-21. In contrast, the 3G8 IgG and 2B1 Bsab both activated the PMN with baseline MFI ratio values of 1.19 and 0.94, respectively, to peak values of 2.44 at 30 s (3G8 IgG) and 1.16 at 70 s. (2B1 Bsab) (Fig. 3Go).



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  		FIGURE 3. Ca2+ flux assay measuring Bsab-mediated leukocyte activation. PBMC and PMN were freshly isolated from heparinized whole blood obtained from healthy donors and pooled. The cells were loaded with 4 µM Fluo-3 and 10 µM Fura Red dyes, and excess dye was removed. The cells were subsequently collected on a flow cytometer at baseline (0-s time point) and at 10-s intervals thereafter following the addition of each Ab at 10 µg/ml final concentrations. Activation was detected by calculating the Fluo-3 MFI:Fura Red MFI ratio at each time point, which is an indicator of intracellular Ca2+ mobilization. This experiment is representative of two experiments comparing all three Abs with MOPC-21 as the negative control, of three performed with 3G8 and 2B1, and of five using 3G8 only.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have created and characterized a series of novel scFv-based Bsab that are specific for both HER2/neu and the Fc{gamma}RIII receptor. It has been previously shown that the bispecific mAb 2B1 had significant clinical toxicities due to the simultaneous cellular engagement of its Fc domain and the anti-Fc{gamma}RIII arm of this Ab (25, 34). The Bsab we describe here lack the Fc domain and hence preclude Bsab-directed leukocyte activation until cross-linking occurs through Bsab bound to the tumor Ag. This property may attenuate toxicities that have limited the dosing and possibly the clinical efficacy of 2B1 and other IgG-based Bsab. It was previously demonstrated that a single 3G8 mAb can activate neutrophils by simultaneously engaging two CD16B molecules and CD32A on the surface of a neutrophil (35). Although the 2B1 Bsab can simultaneously engage both of these receptors on the surface of a neutrophil, Vossebeld et al. (35) reported that this interaction with 2B1 Bsab did not increase the intracellular free Ca2+ concentration in neutrophils. Those findings are at variance with the results reported here. In the currently reported Ca2+ mobilization assay, the neutrophils were cocultured with PBMC. Previously, we have shown that the 2B1 Bsab has the ability to bridge neutrophils and monocytes via CD16B and CD32A (29). When the number of PBMC was reduced by 95%, the neutrophils no longer exhibited an increase in the intracellular free Ca2+ concentration (data not shown). Thus, the 2B1 Bsab may trigger Ca2+ mobilization in neutrophils through monocyte-mediated neutrophil activation.

The Bsab described here differ in affinity for HER2/neu with KD values ranging from 10-7 to 10-10 M. Significantly, the trend in affinity observed with the scFv derivatives remains intact in the Bsab format; the C6G98A-NM3E2 molecule displayed the lowest affinity for HER2/neu followed by C6.5-NM3E2, C6ML3.9-NM3E2, C6B1-NM3E2, and C6B1D2-NM3E2 (in order of increasing affinity). Each molecule bound to the Fc{gamma}RIII receptor with an average affinity of 3.4 x 10-7 M. It is important to note that some of the affinities for HER2/neu were attenuated from those observed with the monomeric scFv format. Most notably, the C6G98A-NM3E2 and the C6B1D2-NM3E2 Bsab experienced the largest change in overall affinity. For both molecules, this modification was caused by increases in off rates. This finding is not surprising given that the molecules doubled in size through the addition of a second domain (the NM3E2 scFv). The joining of two scFvs by a linker caused an apparent shift in the structure of the individual scFvs, leading to a slight diminution in their respective affinities. Although we have not formally examined the mechanisms responsible for these changes, it is reasonable to speculate that these differences result from steric hindrance imposed by the added subunit that reduced the flexibility of each binding domain.

A similar change in affinity between scFv and the Bsab formats was observed with the 4-4-20 (anti-fluorescein) x 4-01 (anti-ssDNA) Bsab. With this particular molecule the association constant (KA) of the 4-4-20 portion of the Bsab was reduced when compared with the association constant of the corresponding scFv (36). Furthermore, the Ucht1v9 x MOC31 anti-human CD3{epsilon} x epithelial gp2 diabody represents another example where changing the format of the molecule resulted in a lower affinity. In this molecule, the MOC31 arm of the diabody exhibited a faster off rate than the MOC31 scFv (37).

Values for the scFv-binding affinities as well as those for the NM3E2 and the C6.5-NM3E2 molecules were previously published (21, 30, 38, 39). However, the numbers shown in Table IGo represent kinetic experiments performed using newer methodologies that likely account for any differences observed between previous experiments and the results shown here. Changes from the previously published methodology include a slight increase in the pH (4.5–5.2) of the solution in which the ligand is suspended in before coating it onto the chip. Although the ligand must be in an acidic buffer to allow for conjugation to the chip, it is necessary to find the highest acidic pH at which enough ligand will bind to protect its structural integrity. Also, in contrast to previous experiments, a control curve was subtracted from the test curves to eliminate background during the analysis. The elimination of background as well as a greater confidence in the structural integrity of the ligand provides a more accurate delineation of the binding affinities. Furthermore, testing a wide range of Bsab concentrations allowed for the determination of the kon and koff values from a set of curves that had been measured under identical conditions.

Cytotoxicity experiments revealed a correlation between an increase in binding affinity for HER2/neu and the ability of the Bsab to augment lysis of SKOV-3 tumor cells. This finding suggests that higher affinity Bsab are retained longer by tumor cells, thus allowing more time for the leukocytes to bind to the available anti-Fc{gamma}RIII binding domain of the Bsab. Studies of other bispecific Abs have also shown a similar positive correlation between an increased Ag affinity and cytotoxic capability (40, 41).

In the current study, we found that although the correlation between affinity and lysis was most prominent at a 50:1 E:T ratio, the same trend was observed at a 25:1 ratio. However, at the 5:1 E:T ratio, the differences in lysis induction between Bsab was attenuated significantly, probably because only modest levels of cytotoxicity were achieved under these conditions. This observation suggests important areas for future improvements in the design of Bsab, because the microenvironment of solid tumors typically contains exceedingly low E:T ratios.

The 2B1-bispecific IgG molecule, which has a lower affinity for HER2/neu than the highest affinity Bsab tested here (KD = 2.3 x 10-8 M for 2B1 vs 1.7 x 10-10 M for C6B1D2-NM3E2), induced peak tumor lysis at lower concentrations than any of the molecules described here. This observation indicates that affinity for the tumor Ag is not the only factor involved in achieving efficient tumor lysis. It is possible that the anti-Fc{gamma}RIII domain of 2B1 (derived from the 3G8 mAb) is a more efficient activator of lysis through this receptor than is NM3E2, which was used to make the molecules tested here. The size of a Bsab molecule also may play a role in its ability to induce antitumor cytotoxicity. The binding arms of the 2B1 Ab may have more flexibility and a larger span than do the (scFv)2 Bsab described in this report. However, it is important to note that the small Bsab molecules described here may exhibit better tumor penetration properties and therefore possess better in vivo therapeutic potential then the large 2B1 IgG Bsab. The lack of an Fc domain should not hinder cytotoxicity potentiation by (scFv)2-based Bsab since 2B1 is an efficient promoter of cytotoxicity in either the IgG or (Fab')2 formats (10).

The results of these studies indicate that numerous factors must be considered in the design of multifunctional Ab-based proteins. Domains that are irrelevant to the intended mechanism of action, such as Fc domains on Bsab, may contribute independent ligand interactions that could be beneficial or detrimental (e.g., leukocyte activation in the absence of tumor cell engagement). The choice of activating ligands is clearly critical, as this determines the leukocyte population to be targeted and defines the type of activation that will ensue. The affinity of Bsab interactions for target ligands has important implications as well. It is reasonable to speculate that the best results will be obtained with Bsab that target leukocytes with low affinity and do not promote their activation in the absence of relevant tumor cell engagement. NM3E2 meets these criteria, though the magnitude of lysis promotion in the (scFv)2 format may be suboptimal. Clearly, affinity for the tumor Ag is a major factor influencing Ab localization and efficacy. We have previously reported that a minimum scFv affinity in the range of 10-8 M is required for measurable tumor retention at 24 h after injection (42). However, it has been hypothesized that Ab molecules with exceedingly high affinity for tumor Ags may suffer from impaired tumor penetration due to the presence of a binding site barrier (43). Recently, we have demonstrated that scFv molecules with very high affinity for HER2/neu exhibit poorer tumor penetration from the vasculature than do their lower affinity variants.7 Accordingly, it may be necessary to balance the competing factors of tumor penetration and efficient promotion of retargeted cytotoxicity to select the appropriate antitumor affinities of Bsab. The results of these studies also suggest a need for the continued refinement of Bsab structures to attain the appropriate span and flexibility for the most efficient possible retargeted lysis. In particular, Bsabs are currently being designed such that certain members of the anti-HER2/neu C6.5 series of scFv will occupy one binding arm of an intact IgG, while the other binding arm will remain specific for Fc{gamma}RIII. Comparison of these molecules with 2B1 as well as with the corresponding (scFv)2 will allow for direct analysis of the effects of and associations between affinity and span and flexibility. A further consideration is the relatively high E:T cell ratios needed to attain significant tumor lysis. Since human tumor milieus will rarely contain E:T ratios that are favorable for ADCC or Bsab-retargeted lysis, Bsab treatment may need to employ leukocyte recruitment strategies to achieve maximal effects.


   Acknowledgments
 
We thank David Ring (Chiron Therapeutics) for kindly providing the 2B1 Bsab and human CD16, Creative Biomolecules for their gift of the HER2/neu Ag, and Dr. Andre Rogatko at the Fox Chase Cancer Center for his statistical expertise.


   Footnotes
 
1 This work was supported by National Institutes of Health Awards CA65559, CA50633, and CA09035-25, by an appropriation from the Commonwealth of Pennsylvania, and by the Frank Strick Foundation and the Bernard A. and Rebecca S. Bernard Foundation. Back

2 Current address: Faculty of Education, University of Melbourne, Victoria, Australia, 3010. Back

3 A.M.M. and L.S. contributed equally to this work. Back

4 Current address: Xerion Pharmaceuticals GmbH, Fraunhoferstrasse 9, D-82152 Martinsried, Germany. Back

5 Address correspondence and reprint requests to Dr. Louis M. Weiner, Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Back

6 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; Bsab, bispecific Ab; scFv, single-chain Fv fragment; PMN, polymorphonuclear neutrophils; fluo-3-AM, fluo-3-acetylmethyl ester; MFI, mean fluorescence intensity; ECD, extracellular domain. Back

7 G. P. Adams, R. Schier, A. M. McCall, H. H. Simmons, E. M. Horak, R. K. Alpaugh, J. D. Marks, and L. M. Weiner. High affinity restricts the localization and tumor penetration of single-chain Fv Ab molecules. Submitted for publication. Back

Received for publication October 30, 2000. Accepted for publication February 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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