|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4750-4757
By
From the Max Delbrück Center for Molecular Medicine,
Berlin-Buch, Germany; Department of Hematology, Oncology, and Tumor
Immunology, Charité-Campus Berlin-Buch, Humboldt University,
Berlin-Buch, Germany; Tumorimmunology Program, German Cancer Research
Center, Heidelberg, Germany.
Bispecific antibodies (CD3x19) against the CD3
BISPECIFIC ANTIBODIES have been generated
to circumvent the problem of antigen recognition in T-cell-based
immunotherapy models. Such antibodies consisting of an
anti-CD3 It is generally accepted that T-cell activation requires two distinct
signals. The first signal depends on the ligation of the T-cell
receptor (TCR)/CD3 complex and the CD4 or CD8 coreceptors.5 The second signal can be provided by cell surface molecules that mediate essential costimulatory signals, thereby complementing the
TCR/CD3-mediated events.6,7 CD28 is such a potent
costimulatory molecule, and ligation of CD28 with agonistic antibodies
or its natural ligands (B7.1 (CD80) and B7.2 (CD86)) synergizes with TCR-mediated signaling to initiate and maintain T-cell
responses.7,8 Recently, ligation of CD28 by agonistic
antibodies has been shown to prevent activation-induced cell death
(AICD) of naive T cells during primary stimulation.9 This
was related to the upregulation of bcl-xL, a potent
apoptosis-preventing member of the bcl-2 gene family.
We previously described that cytotoxic T cells undergo AICD on
target-cell recognition.10 Thus, the cells hit and kill but then die by AICD. This may have importance in limiting potentially dangerous immune responses, eg, in the control of autoimmunity. We
coined the term "veto" apoptosis to describe this phenomenon in
analogy to the classical veto T-cell suppression.11 Veto apoptosis, like other forms of T-cell AICD,12,13 is
mediated by the Fas ligand.10 Costimulation of the T cells
by B7.1 during target-cell recognition could overcome the veto
apoptosis.10 We therefore asked the question whether
targeting a T cell to a B-lymphoma cell by bispecific antibodies as
well induces veto apoptosis and if cytotoxic T-cell (CTL) apoptosis can
be prevented by costimulation via agonistic antibodies against the CD28
antigen. This is of particular importance in view of the ongoing or
planned clinical trials based on T-cell targeting, because such an
event would preferentially lead to T-cell deletion in contrast to
tumor-cell elimination.
Cell culture and preparation of T cells for in vitro and in vivo
assays.
All cells were maintained in 1640 RPMI (Seromed-Biochrom, Hamburg,
FRG), 10% heat-inactivated fetal calf serum (FCS; GIBCO-BRL, Karlsruhe, FRG), 2 mmol/L L-Glutamine (GIBCO-BRL, Karlsruhe, FRG), and
penicilline-streptomycin (Seromed-Biochrom, Hamburg, FRG). T cells were
prepared from peripheral blood by means of Ficoll density-gradient
centrifugation. B cells were removed by depletion with anti-CD19
magnetic activated cell sorting (MACS) magnetic beads
(Miltenyi Biotec, Bergisch Gladbach, FRG). Adherent cells were removed
by plastic adherence for 2 hours at 37°C. The purity of the T cells
was controlled by staining with PE-labelled anti-CD3 (Becton Dickinson,
Heidelberg, FRG) and analyzed on a FACSort (Becton Dickinson). The
purity of the T cells was over 94%. T cells were activated in 75 cm2 culture flasks (Nunc, Copenhagen, Denmark) by
immobilized OKT3 (flasks coated overnight at 10 µg/mL antibody in
phosphate-buffered saline [PBS]) in the presence of the agonistic
anti-CD28 mab 15E8 (1µg/mL). 30 U IL-2 (Chiron, Ratingen, FRG) were
added after 24 hours to maintain and expand the T cells for a further 5 days. The cytotoxicity of T cells was then determined in a standard 4-hour 51Cr release assay against Raji or Nalm-6 targets.
Bispecific antibodies.
CD3x19 Quadroma cells were generated and cultured as
described14 in a hollow-fiber bioreactor (Tecnomara,
Fernwald, FRG). The derived antibody mixture was first purified by
affinity-chromatography on a Protein-A Sepharose CL-4B column
(Pharmacia, Uppsala, Sweden) to remove immunoglobulin G1 (IgG1)
parental antibodies. Subsequently, the eluent was subjected to HPLC
purification on a Bakerbond ABx column (JT Baker Inc, Philippsburg, NJ)
that allowed a separation of the bispecific antibody fraction by means
of a morpholinoethane sulfonic acid/sodium acetate gradient. Purity of
the eluted material was assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing
conditions as described.14
Immunofluorescence.
B7.1 or B7.2-expression on the B-cell lines Raji and Nalm-6 was
determined by direct staining of 106 cells with
phycoerythrin-labeled anti-CD80 or anti-CD86 mab (Becton-Dickinson). Surface fluorescence was determined using a FACSort (Becton-Dickinson). Dead cells were excluded by propidium iodide (PI) staining.
Measurement of apoptosis: Modified cell-cycle analysis.
Activated T cells (106) were exposed to irradiated target B
cells at a 10:1 ratio. After 24 hours the malignant B cells were removed by depletion with anti-CD19 immunomagnetic beads (Dynal, Chantilly, VA). There was a slight reduction of T-cell numbers in the
range of 5% to 10% after the immunomagnetic depletion, but no
significant difference was observed in T-cell recovery as measured by
absolute numbers of CD2+ cells in the cultures induced with
or without the CD3x19 bs ab in the presence or absence of B-lymphoma
cells. The purity of CD2+ cells was above 97% after MACS
separation. These recovered T cells were pelleted, washed with ice-cold
PBS in U-form 96-well plates, and gently resuspended in 300 µL
hypotonic fluorochrome solution (propidium iodide 50 µg/mL, 0.1 mol/L
sodium citrate plus 0.1% Triton X100) as previously
described.15 After overnight incubation at 4°C in the
dark, the propidium-iodide content of the individual nuclei was
measured on a FACSort (Becton-Dickinson). Cell debris was excluded by
raising the forward-scatter threshold adequately. Apoptotic nuclei
displayed a decreased DNA content below the G1 peak, paralleled by an
increase of the side scatter. Anti-CD95 (anti-APO-1 IgG3) or FII23c
F(ab)2 fragments were produced as previously described.16
Anti-B7.1 antibody BB-1 and control-immunoglobulinM(IgM) were purchased
from Pharmingen (San Diego, CA). CD3x19 bs abs were prepared as
described.1
Measurement of apoptosis: Detection of DNA single-strand breaks by
in situ nick translation (ISNT).
To quantify apoptotic cells we used the method as previously described
for detection of DNA single-strand breaks in apoptotic cells.17 Briefly, the cells were fixed with 1%
formaldehyde followed by 70% ethanol. The fixed cells were washed with
nick buffer (50 mmol/L Tris HCl pH 7.8, 5 mmol/L MgCl2, 0.1 mol/L Animal experiments.
Animals (C.B-17 scid/scid mice [SCID]) from our own breeding
colony) were kept and treated in accordance with local animal protection laws. The mice were kept in isolators under gnotobiotic conditions. Food and water were autoclaved. The mice were not subjected
to antibiotic drug treatment. No mouse pathogens were detected. For
serology, sterile sentinel mice were added to the colony. These mice
were serologicaly negative for Sendai, PVM, MVM, Reo3, MHV (Corona),
Theiler's GD VII, Polyoma, K-, and m.-Adeno virus. Leakiness was
determined by measuring mouse IgM and mouse IgG and mice expressing
serum titers above 50 µg/ml IgM or IgG were excluded from the study.
Tumor cells were injected subcutaneously in the inguinal region (Raji,
107 per animal) or intraperitoneally (Nalm-6,
107 per animal). Human T cells were prepared and
preactivated in vitro by immobilized OKT3, soluble anti-CD28, and
subsequent culture in low-dose IL-2 as described above. 107
activated T cells were injected together with the Raji cells subcutaneously or, with a delay of 1 day, intraperitoneally in the
Nalm-6 experiments. Antibodies were injected intraperitoneally together
with the T cells at the following doses: CD3x19 bs ab at 200 µg/animal, monospecific anti-CD3 (OKT3) plus anti-CD19 (HD37) at 100 µg/animal, anti-CD28 (15E8) at 50 µg/animal. Serum half-life of the
antibodies was determined as described18,19 and was 7.4 days for the CD3x19 and 8.1 days for the anti-CD28 mab. Peritoneal
washout was performed by rigorously injecting 10 mL ice-cold PBS into
the peritoneal cavity of sacrificed mice. Contaminating murine cells
were removed via MACS with anti-H2D d mab and
strepatavidin-magnetic beads (Miltenyi); Nalm-6 cells were depleted
simultaneously as described above.
CD28 costimulation is required for tumor-cell elimination by bispecific
antibodies in vivo.
To determine the in vivo antitumor activity of the CD3x19 bispecific
antibodies, we established xenotransplant models of the Raji Burkitt
lymphoma and Nalm-6 pre-B-ALL in SCID mice. Raji cells, when injected
subcutaneously led to locally growing tumors that, at late stages, also
showed lymphoid dissemination to the inguinal, axillar, thoracic, and
abdominal lymph nodes as described.20 Nalm-6 cells were
injected intraperitoneally and rapidly disseminated to liver, bone
marrow and peripheral blood. The mice rapidly developed hind limb
paralysis due to meningeal infiltration and had to be sacrificed. Human
T cells were preactivated in vitro by immobilized OKT3, soluble
anti-CD28 and low-dose IL-2. These T cells were applied together with
the tumor cells s.c. (Raji) or injected intraperitoneally with a delay
of 1 day (Nalm-6). Control mice received (a) in vitro activated T cells
together with the tumor cells without antibodies or (b) T cells plus
the monospecific anti-CD3 and anti-CD19 antibodies
(Fig 1). These mice showed no increased
survival as compared with mice injected with tumor cells alone.
Coinjection (intraperitoneally) of CD3x19 bs ab together with the T
cells led only to a slightly prolonged survival of mice transplanted
with the Nalm-6 lymphoma cells. Nevertheless, 4 of 10 of the mice
injected with Raji cells were cured by injection of the bispecific
antibody plus T cells. In contrast to this limited survival improvement
with the bs ab CD3x19 alone, the coinjection of anti-CD28 with the
bispecific CD3x19 antibody protected all mice injected with Raji (Fig
1a) and 9 of 10 mice that received Nalm-6 from tumor growth and
dissemination (Fig 1b). The surviving mice were maintained for a
further 6 months and remained disease free during this time. In the
above experiments, the T cells were activated in vitro because SCID
mice transplanted intraperitoneally or intravenously with resting human
peripheral blood lymphocytes showed no antitumor response on
anti-CD3x19 injection. This may be due to the limited recirculation and
survival of human lymphocytes in SCID mice.17 In addition,
there was no significant difference observed in survival (or kinetics
of tumor outgrowth) in control mice injected with tumor cells,
monospecific antibodies, and anti-CD28 as compared with those who
received no anti-CD28 (data not shown). This rules out a nonspecific,
CD3x19-independent enhancement of alloreactivity by the anti-CD28 mab
as cause for the antitumor response mediated by CD3x19 plus anti-CD28
combination therapy.
CD3x19 bispecific antibodies induce apoptosis in the
antibody-targeted cytotoxic T cells.
Because we observed in previous experiments that CTLs can undergo
apoptosis on target-cell contact,10 we tested if this was
also the case in T-cell targeting with CD3x19 bs ab. T cells were
activated polyclonally with plastic-immobilized OKT3 mab plus anti-CD28
mab and maintained in IL-2-containing medium for 5 days. This
stimulation induced optimal T-cell proliferation as measured by
3H-thymidine uptake. The addition of anti-CD28 to the
CD3-cross-linking also yields an improved cytotoxicity of the activated
T cells.1 The activated T cells were then exposed to
allogeneic Raji target cells and cytotoxicity against Raji was measured
in a standard 51Cr-release assay. Bispecific CD3x19 ab was
added in solution to the effector/target cell mixture and triggered
specific lysis of the Raji Burkitt lymphoma target cells at
concentrations above 0.05 µg/mL (data not shown). Addition of
anti-CD28 ab to the CD3x19-targeted T-cell/Raji coculture did improve
killing of the Raji target cells. Similar data were obtained with
Nalm-6 as target cell (data not shown). To test if the T cells
themselves underwent activation-triggered apoptosis in this system, we
assessed T-cell apoptosis by measuring nuclear DNA content of the T
cells. A large percentage of the T cells was induced to apoptosis by
the T-cell targeting during coculture with the target cells
(Fig 2). The addition of CD3x19 bispecific
ab to the target cells induced apoptosis in 65% of the T cells when
Nalm-6 was employed as target (Fig 2a) and 30% of the T cells in the
case of Raji target cells (Fig 2b). The addition of anti-CD28 reduced
the amount of T-cell apoptosis in both systems by about 50%.
Veto apoptosis is mediated by the Fas ligand.
The CD95/Fas ligand (FasL) has been implicated in T-cell
AICD.12 We previously observed that AICD of CTLs, which we
termed veto apoptosis, is mediated in part by autocrine production of the FasL by the T cells on target-cell contact.13 The
addition of F(ab)2 fragments of anti-CD95 (anti-APO-1 IgG3) ab could
inhibit T-cell AICD by preventing binding of the FasL to the CD95
antigen.12 We therefore investigated if the target-induced
veto apoptosis is, like other forms of AICD, as well mediated by the
FasL. We used Nalm-6 as target cells, exposed them to activated T cells and added control F(ab)2 or anti-CD95 F(ab)2 to the CD3x19-triggered cultures. Although 61% of the T cells were induced to apoptotic death
by the CD3x19 bs ab in the presence of Nalm-6 targets, the addition of
anti-CD95 F(ab)2 fragments decreased T-cell AICD to 32%. No reduction
was observed with the control F(ab)2 (Fig
4).
CD28 costimulation prevents activation-induced cell death in vivo.
The previous experiments showed that T-cell activation by CD3x19 bs ab
alone leads to AICD and that CD28-mediated costimulation protects from
T-cell AICD in vitro during target-cell contact. In addition, we knew
that costimulation through CD28 drastically improved the antitumor
effect of the CD3x19 bispecific ab in the SCID xenotransplant models.
We therefore assessed whether such a protection from AICD also occurs
in vivo. Because the Nalm-6 model completely depends on anti-CD28 for
tumor prevention we selected it for the in vivo apoptosis assays.
Groups of three SCID mice were injected intraperitoneally with Nalm-6
cells alone or in combination with T cells, CD3x19 bs ab, with or
without anti-CD28. After 16-hour incubation in vivo, the T cells were recovered by peritoneal washout and T-cell apoptosis was determined, after removal of the contaminating murine cells and Nalm-6 cells, by
ISNT staining for DNA strand breaks. The average of background apoptosis of the T cells in the absence of Nalm-6 or antibodies was
9%. When the T cells were injected together with Nalm-6 and the
monospecific control antibodies, the apoptosis rate of the T cells was
8%. In the presence of the CD3x19 antibody and Nalm-6, 30.7% of the T
cells showed apoptotic DNA strand breaks that could be decreased to
14.4% (P < .01) in the presence of anti-CD28 mab (Table 1). The half life of the antibodies
was 7.1 days for the CD3x19 bs ab and 8.2 days for 1.5 E8 anti CD28 mab
(data not shown).
T-cell activation is initiated by signals transmitted through the
T-cell receptor/signal transduction complex (signal 1). Other
costimulatory receptor-ligand interactions between T-cells and
antigen-presenting cells (APC) are needed, however, for complete activation (signal 2). Signaling by APC that provide the antigen signal
in combination with costimulation, leads to activation, proliferation,
and differentiation. Signaling through the T-cell receptor alone can
lead to anergy5 or AICD.10 Previous in vivo
studies showed that the efficacy of T cells against tumor cells in
immunotherapy approaches can be improved by delivering costimulatory
signals. Costimulation by CD28-mediated signals affects a wide variety
of T-cell activation parameters (reviewed in7,8) and can
protect T cells from AICD.9,10 In bispecific antibody-mediated T-cell targeting, immunodeficient mice were cured
from established human tumors when the mice were treated with both the
CD3xCD30 bs ab and received a costimulatory signal via a second
CD28xCD30 bs ab.21 New strategies for costimulation of T
cells to generate anticancer immunity focus on the generation of B7
constructs that may be delivered to the tumor-cell targets, eg, via
(retro-) viral vectors,10 or by injection of chimeric fusion proteins in which the B7.1 or B7.2 is linked by genetical engineering to molecules such as single-chain antibodies22
which then mediate targeting of the costimulatory construct. In this line, the costimulation via agonistic CD28 antibodies was shown to
overcome anergy induced by signal 1 alone and to allow the activation
of primed or preactivated T cells but not necessarily of the naive T
cells in a syngeneic murine model.23 Such a requirement for
costimulation was shown as well in other studies, in which costimulation via B7/CD28 facilitated the generation of CTLs during CD3-redirected cytotoxicity assays in vitro not only from the CD45RO+ memory population but also from small resting
(CD45RO-) T cells.24
We thank R.A.W. van Lier, CLB, Amsterdam, The Netherlands and G. Moldenhauer, German Cancer Research Center, Heidelberg, FRG for the
most generous gift of purified 15E8 anti-CD28 antibody and P.H. Krammer
for kindly providing the anti-APO-1 IgG3 antibody. We are grateful to
C.H. Köhne, University of Rostock, FRG, for helpful discussions.
This work was supported by the European Union in the Biomed2 and the
TMR (training and mobility of researchers) program.
Address correspondence to Peter Daniel, MD, Department of Hematology,
Oncology, and Tumor Immunology, Robert Rössle Klinik,
Charité-Campus Berlin-Buch, Humboldt-University, Lindenberger Weg
80, 13125 Berlin-Buch, Germany; e-mail: pdaniel{at}mdc-berlin.de.
1.
Bohlen H, Manzke O, Patel B, Moldenhauer G, Dorken B, von FV, Diehl V, Tesch H:
Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies.
Cancer Res
53:4310, 1993
2.
Haagen IA, van de Griend R, Clark M, Geerars A, Bast B, de Gast B:
Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).
Clin Exp Immunol
90:368, 1992[Medline]
[Order article via Infotrieve]
3.
Haagen IA, Geerars AJ, de Lau WB, Clark MR, van de Griend RJ, Bast BJ, de Gast BC:
Killing of autologous B-lineage malignancy using CD3 x CD19 bispecific monoclonal antibody in end stage leukemia and lymphoma.
Blood
84:556, 1994
4.
Renner C, Jung W, Sahin U, Denfeld R, C. P, Trümper L, Hartmann F, Diehl V, van Lier R, Pfreundschuh M:
Cure of xenografted human tumors by bispecific monoclonal antibodies and human T cells.
Science
264:833, 1994
5.
Schwartz R:
A cell culture model for T lymphocyte clonal anergy.
Science
248:1349, 1990
6.
Freeman G, Freedman A, Segil J, Lee G, Whitman J, Nadler L:
B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells.
J Immunol
143:2714, 1989[Abstract]
7.
Guinan E, Gribben J, Boussiotis V, Freeman G, Nadler L:
Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity.
Blood
84:3261, 1994
8.
Bluestone J:
New perspectives of CD28-B7-mediated T cell costimulation.
Immunity
2:555, 1995[Medline]
[Order article via Infotrieve]
9.
Boise L, Minn A, Noel P, June C, Accavitti M, Lindsten T, Thompson C:
CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL.
Immunity
3:87, 1995[Medline]
[Order article via Infotrieve]
10.
Daniel PT, Kroidl A, Cayeux S, Bargou R, Blankenstein T, Dörken B:
Costimulatory signals through B7.1/CD28 prevent T-cell apoptosis during target-cell lysis.
J Immunol
159:3808, 1997[Abstract]
11.
Miller R:
An immunological suppressor cell inactivating cytotoxic T-lymphocyte precursor cells recognizing it.
Nature
287:544, 1980[Medline]
[Order article via Infotrieve]
12.
Dhein J, Walczak H, Bäumler C, Debatin K, Krammer P:
Autocrine T cell suicide by APO-1/CD95.
Nature
373:438, 1995[Medline]
[Order article via Infotrieve]
13.
Daniel PT, Oettinger U, Mapara MY, Bommert K, Bargou R, Dörken B:
Activation and activation-induced death of human tonsillar B cells and Burkitt lymphoma cells: Lack of CD95 (Fas/APO-1) ligand expression and function.
Eur J Immunol
27:1029, 1997[Medline]
[Order article via Infotrieve]
14.
Csoka M, Strauss G, Debatin KM, Moldenhauer G:
Activation of T cell cytotoxicity against autologous common acute lymphoblastic leukemia (cALL) blasts by CD3xCD19 bispecific antibody.
Leukemia
10:1765, 1996[Medline]
[Order article via Infotrieve]
15.
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C:
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J Immunol Methods
139:271, 1991[Medline]
[Order article via Infotrieve]
16.
Bargou RC, Bommert K, Weinmann P, Daniel PT, Wagener C, Mapara MY, Dörken B:
Induction of Bax-alpha precedes apoptosis in a human B lymphoma cell line: potential role of the bcl-2 gene family in surface IgM-mediated apoptosis.
Eur J Immunol
25:770, 1995[Medline]
[Order article via Infotrieve]
17.
Daniel PT, Krammer PH:
Activation induces sensitivity towards APO-1 (CD95)-mediated apoptosis in human B cells.
J Immunol
153:5624, 1994
18.
Coney LR, Daniel PT, Sanborn D, Dhein J, Debatin KM, Krammer PH, Zurawski VJ:
Apoptotic cell death induced by a mouse-human anti-APO-1 chimeric antibody leads to tumor regression.
Int J Cancer
58:562, 1994[Medline]
[Order article via Infotrieve]
19.
Dhein J, Daniel PT, Trauth BC, Oehm A, Möller P, Krammer PH:
Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens.
J Immunol
149:3166, 1992[Abstract]
20.
Blase L, Daniel PT, Koretz K, Schwartz-Albiez R, Möller P:
The capacity of human malignant B-lymphocytes to disseminate in SCID mice is correlated with functional expression of the fibronectin receptor alpha 5 beta 1 (CD49e/CD29).
Int J Cancer
60:860, 1995[Medline]
[Order article via Infotrieve]
21.
Renner C, Jung W, Sahin U, Denfeld R, Pohl C, Trumper L, Hartmann F, Diehl V, van LR, Pfreundschuh M:
Cure of xenografted human tumors by bispecific monoclonal antibodies and human T cells.
Science
264:833, 1994
22.
Gerstmayer B, Hoffmann M, Altenschmidt U, Wels W:
Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein.
Cancer Immunol Immunother
45:156, 1997[Medline]
[Order article via Infotrieve]
23.
Demanet C, Brissinck J, De JJ, Thielemans K:
Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation.
Blood
87:4390, 1996
24.
Azuma M, Cayabyab M, Philips JH, Lanier LL:
Requirements for CD28-dependent T cell-mediated cytotoxicity.
J Immunol
150:2091, 1993[Abstract]
25.
Krammer PH, Behrmann I, Daniel PT, Dhein J, Debatin KM:
Regulation of apoptosis in the immune system.
Curr Opin Immunol
6:279, 1994[Medline]
[Order article via Infotrieve]
26.
O'Connell J, O'Sullivan GC, Collins JK, Shanahan F:
The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand.
J Exp Med
184:1075, 1996
27.
Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA:
Fas-ligand-induced apoptosis as a mechanism of immune privilege.
Science
270:1189, 1995
28.
Griffith TS, Yu X, Herndon JM, Green DR, Ferguson TA:
CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance.
Immunity
5:7, 1996[Medline]
[Order article via Infotrieve]
29.
Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke RC:
A role for CD95 ligand in preventing graft rejection.
Nature
377:630, 1995[Medline]
[Order article via Infotrieve]
30.
Lau HT, Yu M, Fontana A, Stoeckert CJ:
Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.
Science
273:109, 1996[Abstract]
31.
Rammensee HG, Fink PJ, Bevan MJ:
Functional clonal deletion of class I-specific cytotoxic T lymphocytes by veto cells that express antigen.
J Immunol
133:2390, 1984[Abstract]
32.
Rammensee H:
Veto function in vitro and in vivo.
Int Rev Immunol
4:668, 1989
33.
Zheng L, Fisher G, Miller RG, Peschon J, Lynch DH, Lenardo MJ:
Induction of apoptosis in mature T cells by tumor necrosis factor.
Nature
377:348, 1995[Medline]
[Order article via Infotrieve]
34.
De Gast GC, Van Houten AA, Haagen IA, Klein S, De Weger RA, Van Dijk A, Phillips J, Clark M, Bast BJ:
Clinical experience with CD3 x CD19 bispecific antibodies in patients with B cell malignancies.
J Hematother
4:433, 1995[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
|
Top
Abstract
Introduction
-chain of the
T-cell-receptor/CD3 complex and the CD19 antigen on B cells can target
polyclonal, nontumor-specific T cells to B lymphoma cells. This induces
T-cell activation, and generation of cytotoxic T cells (CTLs). These
polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse
CD19+ B-lymphoma cells. In a xenotransplant model in
severe combined immunodeficiency deficient (SCID) mice, we
and others observed that CD28 triggering is required for efficient
elimination of B-lymphoma cells and cure from the tumor in addition to
CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab,
induces T-cell death by apoptosis. In blocking experiments we showed
that this "veto" apoptosis is mediated by the CD95/Fas ligand.
Addition of anti-CD28 (signal 2) renders the T cells resistant for veto
apoptosis both in vitro and in vivo. We therefore conclude that the
role of costimulation in immunotherapy with bispecific antibodies or
other T-cell-based immune strategies is not only to facilitate T-cell
activation but also to prevent T-cell deletion by apoptosis.
-part (OKT3) and a tumor-cell recognizing moeity can build
a bridge between a T cell and the tumor cell to induce T-cell
activation by CD3 cross-linking. Previous reports have shown that
bispecific antibodies against the CD3 
-mercaptoethanol, 10 µg/mL bovine serum albumin [BSA]) and
incubated with a mixture of 1.3 µL dATP, dCTP, dGTP each 0.2 mmol/L
(Perkin-Elmer-Cetus, Überlingen, FRG), 1.6 µL nick buffer,
0.3 µL 1mmol/L biotinylated-16-dUTP (Boehringer Mannheim, Mannheim,
FRG) and 1 unit E.coli DNA polymerase I (AGS, Heidelberg, FRG) for 90 minutes at 15°C to complete DNA single-strand breaks. The
biotin-labeled cells were stained with avidin-fluorescein
isothiocyanate (FITC) (cell sorter grade; Camon, Wiesbaden, FRG). The
DNA was stained with 1 µg/mL propidium iodide before analysis on a
FACScan (Becton-Dickinson). Percentage of DNA fragmentation corresponds
with percent green fluorescence in the propidium iodide
(PI)+, eg, nuclei-containing population.
, tumor cells alone; 
,
tumor cells plus monospecific control antibodies (anti-CD19 [HD37]
plus anti-CD3 [OKT3], 100 µg each as single dose); 
, tumor cells
plus CD3x19 (200 µg single dose); 
, tumor cells plus CD3x19 (200 µg single dose) plus anti-CD28 (clone 15E8, 50 µg single dose).
) Nalm-6 cells, CD3x19-induced
T-cell AICD in the presence of Raji cells was nevertheless increased
suggesting that endogenous B7.1 can prevent target-induced T-cell AICD
(Fig 
fragments (FII23c IgG3, 5 µg/mL