[Cancer Research 61, 1846-1848, March 1, 2001]
© 2001 American Association for Cancer Research
Target Cell-restricted Triggering of the CD95 (APO-1/Fas) Death Receptor with Bispecific Antibody Fragments1
Gundram Jung2,
Ludger Grosse-Hovest,
Peter H. Krammer and
Hans-Georg Rammensee
Department of Immunology, Institute for Cell Biology, University of Tübingen, D-72076 Tübingen, Germany [G. J., L. G-H., H-G. R.], and Tumor Immunology Program, Division of Immunogenetics, German Cancer Research Center, D-69120 Heidelberg, Germany [P. H. K.]
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ABSTRACT
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Like many other cell surface receptors, the CD95 (APO-1/Fas) molecule needs to be cross-linked by its physiological ligand or by immobilized or multimeric antibodies to mediate biological activity, that is, induction of apoptotic cell death. Monomeric CD95 antibodies of the IgG2a or IgG1 subtype block rather than induce apoptosis. We report here that such antibodies, hybridized to a second antibody directed against a different target antigen on the same cell, effectively induce apoptosis of the cells if the expression of the target antigen exceeds a certain threshold level. It appears that this effect is due to bicellular binding of bispecific antibodies resulting in mutual cross-linking of the CD95 death receptor and the target antigen. Using bispecific reagents, it may therefore be possible to restrict the activation of death receptors to a given target site, e.g., a tumor. In general terms, our findings illustrate a principle according to which the triggering of a cell surface receptor may be confined to a given target cell using bispecific reagents with target X cell surface receptor specificity.
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Introduction
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Bispecific antibodies directed to a target antigen on tumor cells and to the TCR3
-CD3 complex on T cells have been used in the past to direct the activity of these cells toward tumor cells (reviewed in Ref. 1
). If TCR-CD3 antibodies are hybridized to a second T-cell-associated antigen, synergistic effects may result. Emmrich et al. (2)
reported selective stimulation of CD4+ and CD8+ human T cells after incubation with bispecific antibodies consisting of a nonmitogenic anti-TCR antibody and antibodies to CD4 and CD8, respectively. Roosneck et al. (3)
observed that a bispecific antibody with MHC x CD3 specificity induced effective proliferation of a TH clone, whereas a mixture of both parental antibodies failed to do so. We reported previously that a CD3 x CD28 bispecific antibody fragment (bsFab2) induced proliferation of resting human T cells (4)
. Because anti-CD3-mediated activation usually requires immobilized antibodies, the activation by soluble bispecific antibody constructs containing anti-CD3 was unexpected, and the mechanism by which such constructs effect antibody immobilization remained obscure. In particular, it was unclear (a) whether the second, targeting specificity has to be directed to another signaling molecule to allow for bispecific triggering of the TCR-CD3 complex and (b) whether selective and synergistic bispecific triggering can occur with cell surface receptors other than TCR-CD3.
Like CD3, the CD95 death receptor requires binding of immobilized or multimeric antibodies to be activated effectively. The antibodies originally defining this molecule by inducing apoptosis, anti-Fas (5)
and anti-APO-1 (6)
, were of the IgM or IgG3 subtype, respectively. These isotypes cause antigen cross-linking by multimeric binding and self-aggregation of Fc parts, respectively. IgG1 and IgG2a switch variants of the original anti-APO-1 antibody-induced apoptosis only if cross-linked by antimouse Ig antibodies or protein A (7)
. For the present work, we hybridized the IgG2a anti-APO-1 antibody to a second antibody recognizing a target antigen on the same cell. We found that the resulting bispecific antibody fragments (bsFab2) were capable of inducing effective cell death. This enabled us to confine CD95-mediated apoptosis to predefined target cells and to elucidate the mechanism responsible for CD95 multimerization by soluble bispecific reagents.
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Materials and Methods
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Cells and Antibodies.
All cells were kept in RPMI 1640 supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Sigma, Deisenhofen, Germany). SKW6.4 and Jurkat cells are CD95-expressing, apoptosis-sensitive cells of B- and T-cell origin, respectively. Hybridomas producing monoclonal antibodies directed to CD2 (OKT11), CD5 (OKT1), CD19 (4G7), CD28 (9.3), and CD40 (G-28.5) were supplied by Dr. R. Levy (Stanford, CA), Dr. J. A. Ledbetter (Seattle, WA), and the American Type Culture Collection (Manassas, VA), respectively. The APO-1 antibody and isotype switch variants thereof have been described previously (6
, 7)
. All antibodies were purified from hybridoma supernatants using protein A affinity chromatography, except anti-CD20 antibody L3b3, which was supplied in purified form by Dr. Carsten Brockmeyer (Baxter, Unterschleissheim, Germany). 7C11, an agonistic IgM antibody directed to CD95, was purchased from Immunotech (Marseille, France).
Generation of Bispecific Antibody Fragments.
Bispecific antibody fragments (bsFab2) were prepared by selective reduction and reoxidation of hinge region disulfide bonds as described previously (4)
. The reaction conditions used prevent the formation of homodimers and allow almost complete hybridization of modified Fab fragments of the parental antibodies. For this study, we hybridized the IgG2a variant of the APO-1 antibody to antibodies directed against the B-cell-associated antigens CD19, CD20, and CD40 and to the antigens CD2, CD5, and CD28 expressed on Jurkat cells.
FACS Analysis.
SKW6.4 and Jurkat cells were analyzed for expression of APO-1 and the six target antigens mentioned above after incubation with the respective target antibodies (10 µg/ml) and FITC-labeled antibodies to mouse IgG (Dako, Hamburg, Germany). FACS analysis was performed using a FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA).
Determination of Tumor Cell Killing.
Target cells (SKW6.4 and Jurkat) were incubated in triplicates in 96-well plates (1 x 105 cells/well) with 1 µg/ml respective antibody constructs. After 16 h, viability of the cells was measured using the tetrazolium salt WST-1 (Boehringer Mannheim, Mannheim, Germany), which is cleaved by mitochondrial enzymes to form a dark red formazan. Optical density was measured in an ELISA reader (SpectraMax 340; Molecular Devices, Sunnyvale, CA), and percentage of killed cells was calculated as follows: 
, where Ax and Amax are optical densities generated by tumor cells in the presence and absence of antibodies, respectively, diminished by the optical density of medium containing WST-1. In some experiments, including those assessing bystander lysis, tumor cell death was measured using a standard chromium release assay. To this end, target cells were incubated with 51Cr-labeled sodium chromate (3 MBq/ml, 1 h), washed extensively, and seeded in triplicates in 96-well plates (2 x 104 cells/well). After incubation with antibodies for 16 h, the released radioactivity was counted, and the percentage of tumor cell killing was calculated as 
, where cpmmax is radioactivity release by detergent-treated target cells, and cpmspont is spontaneous release in the absence of antibodies. Tumor cell killing measured in the WST and in the 51Cr release assay corresponded closely if it exceeded 20%. At values below 20%, the WST assay appeared to be more sensitive but also showed more variation between different experiments.
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Results
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Bispecific Antibody Fragments (bsFab2) with CD20 x APO-1 Specificity Selectively Kill CD20+ Lymphoma Cells.
Fig. 1
shows that CD20 x Apo-1 bsFab2 fragments induce effective killing of the B-lymphoblastoid cell line SKW6.4 but not of CD20- Jurkat cells. The fact that both cell lines are sensitive to APO-1-mediated cell death is demonstrated by the fact that they are killed by 7C11, an apoptosis-inducing antibody of the IgM subtype. In addition, Jurkat cells are sensitive to bystander lysis (experiments described below). A mixture of Fab fragments of the two parental antibodies failed to induce significant apoptosis of SKW6.4 cells as did the monospecific, bivalent APO-1 antibody. The latter reagent blocked lysis mediated by CD20 x APO-1 bsFab2.
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Fig. 1. Selective killing of SKW6.4 cells after 16 h of incubation with a bispecific antibody fragment with CD20 x APO-1 specificity. Mean values and SDs of three independent experiments are shown.
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The Degree of Killing Corresponds to the Amount of Antigen Expressed on the Target Cells.
In subsequent experiments, we constructed APO-1-2a-containing bsFab2 fragments directed to the T-cell-associated antigens CD2 and CD5 to induce selective killing of Jurkat cells. We found that the former construct (CD2 x APO-1) induced only marginal killing in some experiments and that the CD5 x APO-1 bsFab2 were completely ineffective. A FACS analysis of SKW6.4 and Jurkat cells revealed that CD2 and CD5 are only weakly expressed on Jurkat cells, whereas the expression of CD20 on SKW6.4 cells, as well as that of the B-cell associated antigens CD19 and CD40, was severalfold higher (mean fluorescence intensity values are shown in Fig. 2
). Construction and evaluation of three additional bsFab2 directed to CD28 on Jurkat cells and to CD19 and CD40 on SKW6.4 cells showed that the degree of killing corresponds to the amount of the target antigen expressed on the cells. The CD20 x APO-1 and CD40 x APO-1 constructs induced complete killing of SKW6.4, whereas the CD19 x APO-1 bsFab2 were less effective. On Jurkat cells, only the CD28 x APO-1 construct was capable of inducing significant cell lysis. In any case, apoptosis was induced only on cells carrying the respective target antigen (Fig. 2)
.
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Fig. 2. Selective killing of SKW6.4 and Jurkat cells by different target antibodies hybridized to the APO-1-2a antibody. Assay time, 16 h. Mean values and SDs of at least three independent experiments are shown. Mean fluorescence intensity (MFI) values for the indicated target antigens are given (one representative experiment of three independent experiments) at the right.
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Triggering of the CD95 Death Receptor Is Associated with Bicellular Binding of Bispecific Antibodies.
Theoretically, a bispecific antibody recognizing two antigens on the same cell can bind in a mono- or bicellular fashion as depicted in Fig. 3
. When 51Cr-labeled Jurkat cells were incubated with SKW6.4 cells and CD20 x APO-1 or CD40 x APO-1 bsFab2, we noticed a profound and almost complete bystander killing of the Jurkat cells (Fig. 4)
. This demonstrates that the bsFab2 bound to SKW6.4 have a free CD95 specificity available for binding to and triggering the CD95 molecule on the bystander cells (Fig. 3C)
. It is unlikely that this phenomenon is induced merely by an excess of CD40 over CD95 because the expression level of both molecules on SKW6.4 cells is comparable (data not shown). Thus, it appears that the target antigen and CD95 are mutually cross-linked by bicellular binding of the bispecific antibody as depicted in Fig. 3B
.
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Fig. 3. In principle, bispecific antibodies directed to two different antigens on the same cell may bind in a monocellular (A) or bicellular (B) fashion. In the latter case, mutual cross-linking of both antigens is induced, and bystander lysis may occur if one of the antigens is the CD95 death receptor, and the bystander cell carries CD95 but not the target antigen (C).
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Fig. 4. Killing of CD20/CD40-negative Jurkat cells by CD20 x APO-1 and CD40 x APO-1-bsFab2 bound to CD20/CD40-positive SKW6.4 cells. 51Cr-labeled Jurkat cells (2.5 x 104 cells/well) were incubated for 16 h with the antibodies indicated in the presence and absence of SKW6.4 cells (7.5 x 104 cells/well). Mean values and SDs of three independent experiments are shown.
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Discussion
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It is well established that certain antibodies directed to the CD95 (APO-1/Fas) death receptor induce apoptosis of CD95-positive and -sensitive cell lines. However, the therapeutic use of such antibodies, e.g., for killing of CD95 expressing tumor cells in vivo, is hampered by an expression of this receptor on normal cells, e.g., on hepatocytes. Thus, it is not surprising that in initial experiments, the application of agonistic CD95 antibodies in mice led to fatal hepatic failure (8)
. Since then, considerable effort has been made to reduce the toxicity of anti-CD95 antibodies in vivo (9
, 10)
. Our results imply that bispecific antibodies with target X APO-1 specificity might be able to confine activation of CD95 and possibly other death receptors to predefined target cells. The magnitude of this effect seems to be correlated to the expression level of the respective target antigen. Nevertheless, other factors such as the microarchitecture of antigens at the cell surface or their capability to modulate on antibody binding may influence the effectivity of target x CD95 bispecific constructs. In addition, if the target antigen is a signaling molecule by itself, it may also be triggered by bispecific antibodies with target x APO-1 specificity. In this respect, it has been reported that immobilized (11
, 12)
or homoconjugated (13)
antibodies directed to CD20 (11
, 13)
and CD40 (12)
may induce apoptosis, whereas CD28 triggering inhibits the apoptosis of T cells, at least under certain experimental conditions (14)
.
In addition to the target antigen selected, the particular CD95 antibody used may influence the effectivity of target x CD95 antibodies. We noted in initial experiments that bsFab2 with CD20 x FasM3 specificity failed to induce effective apoptosis of SKW6.4 cells (data not shown). FasM3 is a CD95 antibody of the IgG1 subtype that is supposedly capable of triggering apoptosis after cross-linking. In conclusion, the effect of target x CD95 bsFab2 may be optimized by choosing a target antigen with a high and stable expression level and a CD95 antibody with an optimal stimulatory capacity.
We and others have previously observed synergistic effects on T-cell activation if antibodies to the TCR-CD3 complex are hybridized to antibodies recognizing a second antigen on the T cells (2, 3, 4)
. Roosneck et al. (3)
attributed the activity of such a construct with MHC x CD3 specificity to monocellular binding of bispecific antibodies, which brings the CD3 complex in close proximity to other membrane molecules, thereby inducing a critical perturbance of membrane dynamics that eventually results in CD3-mediated signaling. Although our results do not rule out such monocellular binding, bystander killing of cells expressing CD95 but not the target antigen clearly indicates that at least in the case of CD95-triggering bicellular binding of bispecific constructs does occur (Fig. 3)
. This means that in principle, any target antigen should allow effective immobilization of anti-CD95 antibodies incorporated in bispecific constructs with target x CD95 specificity. However, as discussed above, this does not rule out a peculiar role of the target antigen, which may support or counteract induction of apoptosis with its own signaling function.
The immunological consequences of apoptotic cell death are controversial at present. The prevailing view that apoptosis is characterized by the absence of an immunological response has been supported by several reports describing the activation of dendritic cells by necrotic but not apoptotic cells (reviewed in Ref. 15
). However it has also been pointed out that at least under certain conditions, potent proinflammatory cytokines may be generated during the activation of the apoptotic cascade and that the transfection of the CD95 ligand into tumor cells induces an inflammatory immune response rather than suppressing it (reviewed in Ref. 16
). This raises the possibility that on in vivo application, bicellular binding of target x APO-1 bispecific antibodies may induce inflammatory responses in a similar way.
Regardless of the mechanisms involved, our results clearly demonstrate that the possibility of selective and synergistic stimulation with bispecific antibodies is not restricted to the TCR-CD3 complex. In fact, it is tempting to extend our findings to all surface receptors that require multimerization by immobilized antibodies or physiological ligands to become activated. Bispecific reagents (not necessarily antibodies) binding to two such surface receptors on the same cell may in effect trigger both receptors, most likely by inducing a mutual cross-link as depicted in Fig. 3B
. This principle may be used to induce simultaneous activation of two different receptors on the same cell and to restrict the stimulation of an important cellular receptor like CD95 to the proximity of a given cell type.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a grant from the Deutsche Forschungsgemeinschaft administered through the Sonderforschungsbereich 510 Stem Cell Biology and Antigen Processing. 
2 To whom requests for reprints should be addressed, at Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany. Phone: 49-7071-29-87621; Fax: 49-7071-29-5653; E-mail: Gundram.Jung{at}uni-tuebingen.de
3 The abbreviations used are: TCR, T-cell receptor; FACS, fluorescence-activated cell-sorting.
Received 12/18/00.
Accepted 1/18/01.
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Cancer Res.,
February 15, 2008;
68(4):
1221 - 1227.
[Abstract]
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ABSTRACT
Introduction
