Target Cell-restricted Triggering of the CD95 (APO-1Fas) Death by lcp19892


									[CANCER RESEARCH 61, 1846 –1848, March 1, 2001]

Advances in Brief

Target Cell-restricted Triggering of the CD95 (APO-1/Fas) Death Receptor with
Bispecific Antibody Fragments1
Gundram Jung,2 Ludger Grosse-Hovest, Peter H. Krammer, and Hans-Georg Rammensee
                                                                     ¨                 ¨
Department of Immunology, Institute for Cell Biology, University of Tubingen, D-72076 Tubingen, 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.]

Abstract                                                                              anti-APO-1 (6), were of the IgM or IgG3 subtype, respectively. These
                                                                                      isotypes cause antigen cross-linking by multimeric binding and self-
   Like many other cell surface receptors, the CD95 (APO-1/Fas) molecule
                                                                                      aggregation of Fc parts, respectively. IgG1 and IgG2a switch variants of
needs to be cross-linked by its physiological ligand or by immobilized or
                                                                                      the original anti-APO-1 antibody-induced apoptosis only if cross-linked
multimeric antibodies to mediate biological activity, that is, induction of
apoptotic cell death. Monomeric CD95 antibodies of the IgG2a or IgG1                  by antimouse Ig antibodies or protein A (7). For the present work, we
subtype block rather than induce apoptosis. We report here that such anti-            hybridized the IgG2a anti-APO-1 antibody to a second antibody recog-
bodies, hybridized to a second antibody directed against a different target           nizing a target antigen on the same cell. We found that the resulting
antigen on the same cell, effectively induce apoptosis of the cells if the            bispecific antibody fragments (bsFab2) were capable of inducing effec-
expression of the target antigen exceeds a certain threshold level. It appears        tive cell death. This enabled us to confine CD95-mediated apoptosis to
that this effect is due to bicellular binding of bispecific antibodies resulting in   predefined target cells and to elucidate the mechanism responsible for
mutual cross-linking of the CD95 death receptor and the target antigen.               CD95 multimerization by soluble bispecific reagents.
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
                                                                                      Materials and Methods
surface receptor may be confined to a given target cell using bispecific                      Cells and Antibodies. All cells were kept in RPMI 1640 supplemented
reagents with target X cell surface receptor specificity.                                  with 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and
                                                                                           10% heat-inactivated fetal bovine serum (Sigma, Deisenhofen, Germany).
Introduction                                                                               SKW6.4 and Jurkat cells are CD95-expressing, apoptosis-sensitive cells of B-
                                                                                           and T-cell origin, respectively. Hybridomas producing monoclonal antibodies
   Bispecific antibodies directed to a target antigen on tumor cells and to directed to CD2 (OKT11), CD5 (OKT1), CD19 (4G7), CD28 (9.3), and CD40
the TCR -CD3 complex on T cells have been used in the past to direct the (G-28.5) were supplied by Dr. R. Levy (Stanford, CA), Dr. J. A. Ledbetter
activity of these cells toward tumor cells (reviewed in Ref. 1). If TCR- (Seattle, WA), and the American Type Culture Collection (Manassas, VA),
CD3 antibodies are hybridized to a second T-cell-associated antigen, respectively. The APO-1 antibody and isotype switch variants thereof have
synergistic effects may result. Emmrich et al. (2) reported selective been described previously (6, 7). All antibodies were purified from hybridoma
stimulation of CD4 and CD8 human T cells after incubation with supernatants using protein A affinity chromatography, except anti-CD20 an-
bispecific antibodies consisting of a nonmitogenic anti-TCR antibody and tibody L3b3, which was supplied in purified form by Dr. Carsten Brockmeyer
antibodies to CD4 and CD8, respectively. Roosneck et al. (3) observed (Baxter, Unterschleissheim, Germany). 7C11, an agonistic IgM antibody di-
that a bispecific antibody with MHC CD3 specificity induced effective rected to CD95, was purchased from Immunotech (Marseille, France).
                                                                                              Generation of Bispecific Antibody Fragments. Bispecific antibody frag-
proliferation of a TH clone, whereas a mixture of both parental antibodies
                                                                                           ments (bsFab2) were prepared by selective reduction and reoxidation of hinge
failed to do so. We reported previously that a CD3 CD28 bispecific
                                                                                           region disulfide bonds as described previously (4). The reaction conditions
antibody fragment (bsFab2) induced proliferation of resting human T used prevent the formation of homodimers and allow almost complete hybrid-
cells (4). Because anti-CD3-mediated activation usually requires immo- ization of modified Fab fragments of the parental antibodies. For this study, we
bilized antibodies, the activation by soluble bispecific antibody constructs hybridized the IgG2a variant of the APO-1 antibody to antibodies directed
containing anti-CD3 was unexpected, and the mechanism by which such against the B-cell-associated antigens CD19, CD20, and CD40 and to the
constructs effect antibody immobilization remained obscure. In particu- antigens CD2, CD5, and CD28 expressed on Jurkat cells.
lar, it was unclear (a) whether the second, targeting specificity has to be                   FACS Analysis. SKW6.4 and Jurkat cells were analyzed for expression of
directed to another signaling molecule to allow for bispecific triggering APO-1 and the six target antigens mentioned above after incubation with the
of the TCR-CD3 complex and (b) whether selective and synergistic respective target antibodies (10 g/ml) and FITC-labeled antibodies to mouse
bispecific triggering can occur with cell surface receptors other than 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)
   Like CD3, the CD95 death receptor requires binding of immobilized or were incubated in triplicates in 96-well plates (1 105 cells/well) with 1
multimeric antibodies to be activated effectively. The antibodies origi-                     g/ml respective antibody constructs. After 16 h, viability of the cells was
nally defining this molecule by inducing apoptosis, anti-Fas (5) and measured using the tetrazolium salt WST-1 (Boehringer Mannheim, Mann-
                                                                                          heim, Germany), which is cleaved by mitochondrial enzymes to form a
   Received 12/18/00; accepted 1/18/01.                                                   dark red formazan. Optical density was measured in an ELISA reader
   The costs of publication of this article were defrayed in part by the payment of page  (SpectraMax 340; Molecular Devices, Sunnyvale, CA), and percentage of
charges. This article must therefore be hereby marked advertisement in accordance with
                                                                                          killed cells was calculated as follows: (1 Ax/Amax) 100, where Ax and
18 U.S.C. Section 1734 solely to indicate this fact.
     Supported in part by a grant from the Deutsche Forschungsgemeinschaft administered   Amax are optical densities generated by tumor cells in the presence and
through the Sonderforschungsbereich 510 Stem Cell Biology and Antigen Processing.         absence of antibodies, respectively, diminished by the optical density of
     To whom requests for reprints should be addressed, at Department of Immunology,      medium containing WST-1. In some experiments, including those assessing
                ¨                                              ¨
University of Tubingen, Auf der Morgenstelle 15, D-72076 Tubingen, Germany. Phone:
                                                                                          bystander lysis, tumor cell death was measured using a standard chromium
49-7071-29-87621; Fax: 49-7071-29-5653; E-mail:
     The abbreviations used are: TCR, T-cell receptor; FACS, fluorescence-activated       release assay. To this end, target cells were incubated with 51Cr-labeled
cell-sorting.                                                                              sodium chromate (3 MBq/ml, 1 h), washed extensively, and seeded in triplicates
                                                       RESTRICTED TRIGGERING OF CD95 WITH BISPECIFIC ANTIBODIES

in 96-well plates (2 104 cells/well). After incubation with antibodies for 16 h,
the released radioactivity was counted, and the percentage of tumor cell killing was
calculated as cpm        cpmspont/cpmmax       cpmspont      100, 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.


   Bispecific Antibody Fragments (bsFab2) with CD20                 APO-1
Specificity Selectively Kill CD20 Lymphoma Cells. Fig. 1 shows that
CD20       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 dem-
onstrated 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 frag-
ments 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 APO-1 bsFab2.
                                                                                             Fig. 2. Selective killing of SKW6.4 and Jurkat cells by different target antibodies
   The Degree of Killing Corresponds to the Amount of Antigen                             hybridized to the APO-1-2a antibody. Assay time, 16 h. Mean values and SDs of at least
Expressed on the Target Cells. In subsequent experiments, we                              three independent experiments are shown. Mean fluorescence intensity (MFI) values for
constructed APO-1-2a-containing bsFab2 fragments directed to the                          the indicated target antigens are given (one representative experiment of three independent
                                                                                          experiments) at the right.
T-cell-associated antigens CD2 and CD5 to induce selective killing of
Jurkat cells. We found that the former construct (CD2              APO-1)
induced only marginal killing in some experiments and that the
CD5 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       APO-1 and CD40           APO-1 constructs induced complete
killing of SKW6.4, whereas the CD19             APO-1 bsFab2 were less
effective. On Jurkat cells, only the CD28          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).
   Triggering of the CD95 Death Receptor Is Associated with
Bicellular Binding of Bispecific Antibodies. Theoretically, a bispe-
cific antibody recognizing two antigens on the same cell can bind in

                                                                                             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).

                                                                                          a mono- or bicellular fashion as depicted in Fig. 3. When 51Cr-labeled
                                                                                          Jurkat cells were incubated with SKW6.4 cells and CD20 APO-1
                                                                                          or CD40      APO-1 bsFab2, we noticed a profound and almost com-
                                                                                          plete 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 ex-
                                                                                          pression level of both molecules on SKW6.4 cells is comparable (data
                                                                                          not shown). Thus, it appears that the target antigen and CD95 are
   Fig. 1. Selective killing of SKW6.4 cells after 16 h of incubation with a bispecific
antibody fragment with CD20        APO-1 specificity. Mean values and SDs of three        mutually cross-linked by bicellular binding of the bispecific antibody
independent experiments are shown.                                                        as depicted in Fig. 3B.
                                                      RESTRICTED TRIGGERING OF CD95 WITH BISPECIFIC ANTIBODIES

                                                                                         target 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 con-
                                                                                         troversial at present. The prevailing view that apoptosis is character-
                                                                                         ized 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
   Fig. 4. Killing of CD20/CD40-negative Jurkat cells by CD20            APO-1 and       tumor cells induces an inflammatory immune response rather than
CD40 APO-1-bsFab2 bound to CD20/CD40-positive SKW6.4 cells. 51Cr-labeled Jurkat
cells (2.5 10 cells/well) were incubated for 16 h with the antibodies indicated in the   suppressing it (reviewed in Ref. 16). This raises the possibility that on
presence and absence of SKW6.4 cells (7.5    104 cells/well). Mean values and SDs of     in vivo application, bicellular binding of target     APO-1 bispecific
three independent experiments are shown.                                                 antibodies may induce inflammatory responses in a similar way.
                                                                                            Regardless of the mechanisms involved, our results clearly dem-
Discussion                                                                               onstrate that the possibility of selective and synergistic stimulation
                                                                                         with bispecific antibodies is not restricted to the TCR-CD3 complex.
   It is well established that certain antibodies directed to the CD95                   In fact, it is tempting to extend our findings to all surface receptors
(APO-1/Fas) death receptor induce apoptosis of CD95-positive and                         that require multimerization by immobilized antibodies or physiolog-
-sensitive cell lines. However, the therapeutic use of such antibodies,                  ical ligands to become activated. Bispecific reagents (not necessarily
e.g., for killing of CD95 expressing tumor cells in vivo, is hampered                    antibodies) binding to two such surface receptors on the same cell
by an expression of this receptor on normal cells, e.g., on hepatocytes.                 may in effect trigger both receptors, most likely by inducing a mutual
Thus, it is not surprising that in initial experiments, the application of               cross-link as depicted in Fig. 3B. This principle may be used to induce
agonistic CD95 antibodies in mice led to fatal hepatic failure (8).                      simultaneous activation of two different receptors on the same cell
Since then, considerable effort has been made to reduce the toxicity of                  and to restrict the stimulation of an important cellular receptor like
anti-CD95 antibodies in vivo (9, 10). Our results imply that bispecific                  CD95 to the proximity of a given cell type.
antibodies with target X APO-1 specificity might be able to confine
activation of CD95 and possibly other death receptors to predefined                      References
target cells. The magnitude of this effect seems to be correlated to the                  1. Segal, D. M., Weiner, G. J., and Weiner, L. M. Bispecific antibodies in cancer
expression level of the respective target antigen. Nevertheless, other                       therapy. Curr. Opin. Immunol., 11: 558 –562, 1999.
factors such as the microarchitecture of antigens at the cell surface or                  2. Emmrich, F., Rieber, P., Kurrle, R., and Eichmann, K. Selective stimulation of human
                                                                                             T lymphocyte subsets by heteroconjugates of antibodies to the T cell receptor and to
their capability to modulate on antibody binding may influence the                           subset-specific differentiation antigens. Eur. J. Immunol., 18: 645– 648, 1988.
effectivity of target CD95 bispecific constructs. In addition, if the                     3. Roosneck, E., Tunnacliffe, A., and Lanzavecchia, A. T cell activation by a bispecific
                                                                                             anti-CD3/anti-major histocompatibility complex class I antibody. Eur. J. Immunol.,
target antigen is a signaling molecule by itself, it may also be triggered                   20: 1393–1396, 1990.
by bispecific antibodies with target            APO-1 specificity. In this                4. Jung, G., Freimann, U., von Marschall, Z., Reisfeld, R. A., and Wilmanns, W. Target
respect, it has been reported that immobilized (11, 12) or homocon-                          cell induced T cell activation with bi- and trispecific antibody fragments. Eur.
                                                                                             J. Immunol., 21: 2431–2435, 1991.
jugated (13) antibodies directed to CD20 (11, 13) and CD40 (12) may                       5. Yonehara, S., Ishii, A., and Yonehara, M. A cell killing monoclonal antibody
induce apoptosis, whereas CD28 triggering inhibits the apoptosis of T                        (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor
cells, at least under certain experimental conditions (14).                                  necrosis factor. J. Exp. Med., 169: 1747–1756, 1989.
                                                                                          6. Trauth, B. C., Clas, C., Peters, A. M. J., Matzku, S., Moller, P., Falk, W., Debatin,
   In addition to the target antigen selected, the particular CD95 antibody                  K. M. and Krammer, P. H. Monoclonal antibody mediated tumor regression by
used may influence the effectivity of target CD95 antibodies. We noted                       induction of apoptosis. Science (Washington DC), 245: 301–305, 1989.
                                                                                          7. Dhein, J., Daniel, P. T., Trauth, B. C., Oehm, A., Moller, P., and Krammer, P. H.
in initial experiments that bsFab2 with CD20 FasM3 specificity failed                        Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is
to induce effective apoptosis of SKW6.4 cells (data not shown). FasM3                        dependent on cross linking of APO-1 cell surface antigens. J. Immunol., 149:
is a CD95 antibody of the IgG1 subtype that is supposedly capable of                         3166 –3173, 1992.
                                                                                          8. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T.,
triggering apoptosis after cross-linking. In conclusion, the effect of tar-                  Kitamura, Y., Itoh, N., and Nagata, S. Lethal effect of the anti-Fas antibody in mice.
get CD95 bsFab2 may be optimized by choosing a target antigen with                           Nature (Lond.), 364: 806 – 809, 1993.
a high and stable expression level and a CD95 antibody with an optimal                    9. Feng, G., and Kaplowitz, N. Colchicine protects mice from the lethal effect of an
                                                                                             agonistic anti-Fas antibody. J. Clin. Investig., 105: 329 –339, 2000.
stimulatory capacity.                                                                    10. Ichikawa, K., Yoshida-Kato, H., Ohtsuki, M., Oshumi, J., Yamaguchi, J., Takahashi,
   We and others have previously observed synergistic effects on                             S., Tani, Y., Watanabe, M., Shiraishi, A., Nishioka, K., Yonehara, S., and Serizawa,
                                                                                             N. A novel murine anti-human Fas mAb which mitigates lymphadenopathy without
T-cell activation if antibodies to the TCR-CD3 complex are hybrid-                           hepatotoxicity. Int. Immunol., 12: 552–562, 2000.
ized to antibodies recognizing a second antigen on the T cells (2– 4).                   11. Shan, D., Ledbetter, J. A., and Press, O. W. Apoptosis of malignant human B cells by
Roosneck et al. (3) attributed the activity of such a construct with                         ligation of CD20 with monoclonal antibodies. Blood, 91: 1644 –1652, 1998.
                                                                                         12. Schattner, E. J., Elkon, K. B., Yoo, D. H., Tumang, J., Krammer, P. H., Crow, M. K.,
MHC         CD3 specificity to monocellular binding of bispecific anti-                      and Friedman, S. M. CD40 ligation induces APO-1/Fas expression on human B
bodies, which brings the CD3 complex in close proximity to other                             lymphocytes and facilitates apoptosis through the APO-1/Fas pathway. J. Exp. Med.,
membrane molecules, thereby inducing a critical perturbance of mem-                          182: 1557–1565, 1995.
                                                                                         13. Ghetie, M. A., Podar, E. M., Ilgen, A., Gordon, B. E., Uhr, J. W., and Vitetta, E. S.
brane dynamics that eventually results in CD3-mediated signaling.                            Homodimerization of tumor-reactive monoclonal antibodies markedly increases their
Although our results do not rule out such monocellular binding,                              ability to induce growth arrest or apoptosis of tumor cells. Proc. Natl. Acad. Sci. USA,
                                                                                             94: 7509 –7514, 1997.
bystander killing of cells expressing CD95 but not the target antigen                    14. Boise, L. H., Noel, P. J., and Thompson, C. B. CD28 and apoptosis. Curr. Opin.
clearly indicates that at least in the case of CD95-triggering bicellular                    Immunol., 7: 620 – 625, 1995.
binding of bispecific constructs does occur (Fig. 3). This means that                    15. Schild, H. J., and Rammensee, H. G. Gp96, the immune system’s Swiss army knife.
                                                                                             Nat. Immunol., 1: 100 –101, 2000.
in principle, any target antigen should allow effective immobilization                   16. Restifo, N. P. Building better vaccines: how apoptotic cell death can induce inflammation
of anti-CD95 antibodies incorporated in bispecific constructs with                           and activate innate and adaptive immunity. Curr. Opin. Immunol., 12: 597– 603, 2000.


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