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*
Clinical Cooperation Group "Bispecific Antibodies," Department of Otorhinolaryngology, Ludwig-Maximilians-University, Munich, Germany; and
Institutes of Clinical Molecular Biology and Tumor Genetics and Experimental Hematology, Gesellschaft für Strahlung und Umweltforschung-National Research Center for Environment and Health, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R+ accessory cells via its Fc region. We
demonstrate here that the activation of both T lymphocytes and
accessory cells leads to production of immunomodulating cytokines like
IL-1
, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb
elicits excellent antitumor activity in vitro even without the addition
of exogenous IL-2, and therefore represents a totally self-supporting
system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bispecific Abs (bsAb)3
are regarded as powerful tools for the immunological treatment of
malignant cells in a minimal residual disease situation, because single
disseminated tumor cells are especially appropriate targets for an
immunological attack. However, the bsAb described to date normally
activate only a single class of effector cell, i.e., either T cells, NK
cells, FcRI+, or
Fc
RI+ cells (5, 6, 7) following
binding to an appropriate target molecule of the effector cell. Here we
show data obtained with a new class of bsAb consisting of the two
potent and evolutionary related effector subclasses, mouse IgG2a and
rat IgG2b. This intact bsAb (BiUII) is able to simultaneously activate
T cells (via one arm) and accessory cells (via the Fc region) in the
vicinity of tumor cells. In contrast to a similar T cell-redirecting
bsAb, SHR-1, with the subclass combination mouse IgG1 x rat IgG2b
(8), BiUII does not depend on the addition of exogenous
IL-2 to provide full antitumor activity. This reveals the importance of
the subclass combination for induction of activation signals via the Fc
region of accessory cells. Moreover, we demonstrate that the antitumor
efficiency of our bsAb is strongly reduced when T cells or accessory
cells alone are used as effector cells. We therefore postulate that a
"Tri-cell-complex" consisting of tumor cells, T lymphocytes, and
accessory cells is created by this new class of bsAb. Only the
formation of this complex results in a full activation of different
effector cells providing optimal antitumor efficiency.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fadu (American Type Culture Collection, Manassas, VA) and PCI-1 are Ep-CAM-positive established squamous carcinoma cell lines of the head and neck (SCCHN) and were maintained in DMEM with 10% FCS. Both cell lines express epithelial cell adhesion molecule (Ep-CAM) and MHC class I, but not MHC class II as tested by flow cytometry (data not shown). DG75 is an EBV-negative Burkitt lymphoma cell line. PBMC were isolated from heparinized blood of voluntary donors by Ficoll density centrifugation. Where indicated (=PBL), the monocyte/macrophage fraction was removed by adhesion to plastic flasks twice for 2 h at 37°C in an incubator.
Monoclonal Abs
The following hybridomas have been used: 26II6 (rat IgG2b, anti-CD3; kindly provided by R. Schuh, Gesellschaft für Strahlung und Umweltforschung (GSF), Munich, Germany) and C215 (mouse IgG2a, anti-Ep-CAM; kindly provided by M. Dohlsten, Pharmacia Upjohn, Uppsala, Sweden).
RT-PCR
Total RNA from primary lymphocytes was isolated after up to
72 h of incubation with allogeneic SCCHN. The RNA preparations
were treated with RNase-free DNase (Boehringer Mannheim, Mannheim,
Germany) for 30 min at 37°C. After inactivation of the enzyme, 1 µg
RNA was reverse transcribed (Superscript Plus, Life Technologies,
Gaithersburg, MD) with an oligo(dT) primer for 60 min at 42°C. PCR
with one-tenth of the volume (2 µl) was performed in a buffer
containing 1.5 mM MgCl2, 100 pmol of each primer, 0.2 mM
final concentration of each dNTP, and 0.5 µl Goldstar Taqpolymerase (Eurogentec, Seraing, Belgium) in a final volume of
50 µl in an Perkin-Elmer (Norwalk, CT) thermal cycler. PCR primers
are shown in Table I
. Amplified bands
were analyzed by electrophoresis in a 1.5% agarose gel and by ethidium
bromide staining.
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The adherent fraction of PBMC was incubated for 7 days in Iscove’s medium with 5% FCS (Life Technologies) and 800 U/ml of each human IL-4 and GM-CSF (both Boehringer Mannheim).
FACS analysis
For FACS analysis, 105 cells were incubated with the primary Ab for 30 min on ice in PBS/5% FCS. The cells were washed twice in PBS and incubated for another 30 min with the second FITC-labeled Ab. After two final washings, propidiumiodide was added, and flow cytometry was performed using a FACSCalibur cytometer and the CellQuest analysis program (Becton Dickinson, Heidelberg, Germany). For isolation of highly purified CD2+ cells, PBMC were incubated with FITC-labeled Abs (PharMingen, Hamburg, Germany) and separated on a FACSCalibur.
Production of BiUII
The BiUII quadroma was produced as previously described (9). To isolate hybrid Ab molecules of the subclass combination rat IgG2b/mouse IgG2a from hybridoma supernatants, the supernatants were centrifuged, filtered, and loaded onto a 5 ml Econo Pac protein A column (Bio-Rad, Richmond, CA). After washing with 10 volumes of PBS, Ab with the hybrid heavy chain configuration was eluted with 0.1 M citric acid (pH 5.1).
Cell culture and killing efficiency
For determination of bsAb-mediated killing of tumor cells and cytokine production, 1 x 104/well SCCHN (targets, =T) were pipetted in 96-well flat-bottom plates (Falcon, Franklin Lakes, NJ), and PBMC or subpopulations of these effectors (=E) were added at ratios from 40:1 to 1:1 E:T. bsAb was used at 10 ng/well in a total volume of 100 µl/well RPMI with 10% FCS. Plates were incubated for 3 days at 37°C in a humidified atmosphere and 5% CO2. All SCCHN were proven to express Ep-CAM and to lack costimulatory molecules CD80 and CD86.
MTT assay
To assess bsAb-mediated tumor cell killing, a colorimetric MTT-based assay was performed as described previously (10). Briefly, SCCHN target cells were plated in wells of a 96-well flat-bottom plate and incubated overnight to prepare semiconfluent cell monolayers. Effector cells were added to the tumor cell monolayers at the appropriate ratios, and plates were incubated for 24–48 h. After removing effector cells by washing, MTT solution (0.5 mg/ml; Sigma, Deisenhofen, Germany) was added, and plates were incubated for a further 4 h. The MTT solution was removed, and blue crystals of formazan formed in viable tumor cells were dissolved by adding DMSO. Plates were read at 540 nm in a spectrophotometer, and the results were calculated based on the mean of absorbance obtained from at least six wells according to the following formula: % cell death = 100 x (C - E)/(C - B), where C is the optical density reading of the cells with target cells in the absence of effectors (control), B is background without any cell population, and E is the optical density reading of adherent tumor cells remaining in the wells after coincubation with effector cells.
Bioassays
Biologically active levels of IL-2, IL-6, TNF, and GM-CSF were
measured in 96-well flat-bottom microtiter plates using cytokine
dependent growth of cell lines CTLL-2, 7TD1, WEHI-164, and TF-1,
respectively. Cell culture supernatants were titrated in duplicates and
diluted from 1:5 to 1:10000. Standards of recombinant human IL-2 (PBH,
Hannover, Germany), human IL-6 (Boehringer Mannheim), human TNF-
(PBH), or human GM-CSF (Boehringer Mannheim) were included in each
assay to generate a standard curve. Intra- or interassay variability
was less than 10% or 20%, respectively. Specificity of bioassays was
confirmed by neutralizing active samples with cytokine specific Abs
(PBH and Boehringer Mannheim). The lower detection limits of IL-2,
IL-6, TNF, and GM-CSF were 20 pg/ml, 10 pg/ml, 0.1 ng/ml, and 10 pg/ml,
respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ep-CAM is an Ag that is overexpressed on many carcinomas of
different origin (11). Therefore, it was selected as a
target tumor molecule for our bsAb. Because T cells are believed to be
the most important effector cells for tumor cell elimination, they were
targeted for activation via CD3. The bsAb constructed to recognize
these two Ags was designated BiUII. Because most tumor cells do not
express costimulatory molecules, BiUII was constructed with mAbs of
subclasses that bind and activate human FcRI+
cells (12). The quadroma that resulted from the fusion of
the anti-Ep-CAM and the anti-CD3 hybridomas was characterized
and intact bsAb was purified as described (9). As shown in
Fig. 1, BiUII represents a chimeric
molecule consisting of the evolutionary related and highly homologous
mouse IgG2a and rat IgG2b heavy chains.
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To determine the antitumor efficiency of BiUII, we first evaluated
its capacity to mediate the killing of the Ep-CAM-positive tumor cell
line PCI-1 (13) and compared it with the two monoclonal
parental Abs (CD3 and Ep-CAM). PCI-1 cells were cocultivated with
PBMC in the presence of either BiUII, both parental Abs, or for control
purposes, without Ab. After 2 days of culture, the numbers of remaining
tumor cells were determined in a standard MTT assay. As shown in Fig. 2, BiUII displayed a much higher lytic
capacity for tumor cells than equimolar concentrations of the
corresponding parental Abs. Even 10-fold higher concentrations (50
ng/100 µl) of these Abs were less efficient than BiUII (data not
shown). Theoretically, the two parental Abs also recruit
FcR+ cells and activate T cells via CD3.
However, their observed antitumor capacity was much lower. This result
suggested that BiUII-mediated formation of a complex involving at least
two different classes of immune cells.
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Induction of ADCC by BiUII
Ab-dependent cellular cytotoxicity (ADCC) is considered to be one
of the most effective mechanisms for the destruction of virally
infected cells and tumor cells (14). Both macrophages and
DC express the high affinity FcRI (CD64) (15) and can
be stimulated to ADCC by particular subclasses of opsonizing IgG Abs.
Because BiUII is a chimeric molecule consisting of a rat IgG2b chain
and a murine IgG2a chain (Fig. 1
), it can theoretically bind and
activate FcRI+ cells (12), a
property that may contribute to BiUII-mediated tumor cell killing. It
has been demonstrated recently that the unconjugated anti-Ep-CAM
17–1A mAb has a therapeutic effect that is probably due to ADCC
(16, 17). To determine whether BiUII could induce ADCC, we
isolated macrophages and DC from peripheral blood by plastic adhesion
and cocultured them with PCI-1 target cells with or without BiUII. The
induction of ADCC measured in a MTT assay clearly demonstrated that
PCI-1 cells were lysed much more efficiently in the presence of BiUII
(Fig. 3). Because T lymphocytes were not
present in this assay, as determined by FACS analysis (data not shown),
the lysis of PCI-1 cells was most likely due to BiUII-mediated
ADCC.
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Because BiUII targets T lymphocytes and
FcRI+ cells, both of which are activated by Ab
binding, we questioned whether accessory cells contributed to the
BiUII-mediated antitumor activity for example via direct phagocytosis.
Therefore, we compared the killing capacity of unseparated PBMCs with
highly purified T cell populations. CD2+ T cells
were isolated by cell sorting and used in a MTT assay. We compared this
purified T cell population with whole PBMC for killing activity
directed against PCI-1 cells. As shown in Fig. 4, maximal antitumor activity was
achieved when PBMCs were used as the effector source of cells. This
result substantiated the importance of accessory cells targeted via
their Fc receptors in tumor cell killing.
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Stimulation of T lymphocytes leads to IL-2 production and
up-regulation of CD25, the -chain of the IL-2R. IL-2 is the most
important autocrine growth factor for T cells (18). In
addition, activated T cells induce IL-12 production by
monocytes/macrophages and DC (19), thereby enhancing
antitumor activity (20).
The high affinity FcRI (CD64) is expressed by monocytes/macrophages
and dendritic cells (15). Because BiUII combines mouse
IgG2a and rat IgG2b subclasses, it should theoretically be able to bind
and activate FcRI+ cells (12).
Therefore, tumor cells opsonized by BiUII are complexed to accessory
cells via CD64 and ADCC may be induced via direct phagocytosis.
Subsequently, phagocytosed tumor-derived proteins can be processed and
presented by MHC class I and II molecules, leading to humoral and
cellular immunity. In addition, activated accessory cells can deliver
cytokines like IL-6 and costimulatory signals via molecules like CD40,
LFA-3, CD80, and CD86 that are mandatory for T cell activation and
prevention of anergy (21).
Therefore, we determined whether BiUII was able to induce the
production of IL-2 and IL-6 in PBMCs when cocultured in the presence of
PCI-1 cells, thereby documenting the activation of both T cells and
accessory cells. As shown in Fig. 5,
PBMCs produce both cytokines only when BiUII is present. IL-2, which is
mainly T cell derived, is produced in essentially equal amounts using
either PBMCs or PBLs as effector cells. In contrast, IL-6 is mainly
produced by activated monocytes/macrophages and was only detectable
when PBMCs were used but was not present when the adherent fraction was
depleted. This finding supports our contention that the Fc region of
BiUII mediates activation of FcR1+ cells.
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, IL-2, IL-4, IL-6, and IL-12. As depicted in Fig. 6, cytokine levels were only slightly
elevated compared with background (IL-2 and IL-12), or were not induced
at all (IL-1, IL-4, and IL-6) when the parental Abs were used as
stimulators. In contrast, after incubation with the bsAb, we observed a
clear induction of all cytokines examined. These data and the fact that
BiUII is much more efficient in tumor cell killing than the parental
Abs suggest that the local production of various cytokines is likely a
prerequisite for efficient tumor cell killing.
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The major drawback in the in vivo application of bsAb is the
problem of side effects provoked by uncontrolled cytokine release. In
particular, the nonspecific activation of T cells via CD3 is thought to
produce elevated IL-2 levels that can cause severe side effects. To
evaluate IL-2 induction by BiUII, PBMCs were incubated with the Burkitt
lymphoma cell line DG75 that does not express Ep-CAM, or with a
subclone of DG75 that was stably transfected with an expression plasmid
for Ep-CAM (DG75/Ep-CAM). As shown in Fig. 7, significant concentrations of IL-2
were only produced when PBMC were cultivated with DG75/Ep-CAM but not
with DG75 cells. This finding implies that full activation of T cells
and production of IL-2 is only achieved as part of a Tri-cell-complex
involving accessory cells and tumor cells. A dependence on the presence
of tumor cells expressing the target Ag Ep-CAM is an important
parameter that can limit the risk of uncontrolled systemic IL-2
production by this bispecific reagent.
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DC are key regulators of immune responses. They form a system of efficient Ag presenting cells which present Ags to T cells (22) and trigger their activation via the CD40 dependent pathway (23). Recently, a DC-specific cytokine, DC-CK1, has been identified that is exclusively expressed by DC at high levels at sites of immune responses (24). DC-CK1 elicits a profound chemotactic activity to attract CD45RA+ naive T cells. Thus it is believed that DC are directly involved in the generation of cytolytic T lymphocytes (23, 25).
Because DC have recently been shown to express CD64 (15),
we wanted to determine whether peripheral blood DC contribute to
BiUII-mediated killing of tumor cells. The adherent fraction of PBMCs
was incubated for 7 days in the presence of IL-4 and GM-CSF and then
cocultured with PCI-1 cells with or without BiUII. Total RNA was then
isolated at two different time points. Whereas after 4.5 h of
incubation no substantial differences in DC-CK1 levels were detectable,
the expression of the cytokine was clearly higher in the presence of
BiUII 16 h later, indicating bsAb-mediated activation of DC (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI+ cells, such as monocytes/macrophages and
DC. This was demonstrated by the induced production of IL-1, IL-2,
IL-6, IL-12, and DC-CK1 (Figs. 6 and 8). Obviously, this strong
activation potential is correlated to the subclass combination mouse
IgG2a x rat IgG2b that, in contrast to other reported
combinations (e.g., mouse IgG2a x mouse IgG1 or rat IgG2b x
mouse IgG1) (26), not only binds but also activates
accessory cells. Although T lymphocytes are believed to be the most important class of immune cell for the elimination of tumor cells, their activation depends on the presence of certain cytokines (most important IL-2) and so-called costimulatory molecules that are usually not delivered by tumor cells themselves. Rather, T cell activation depends on proper Ag presentation of tumor cell-derived peptides by professional APCs that have been demonstrated recently to be required for the induction of a long-lasting tumor immunity (27). New data corroborate the importance of costimulatory molecules for the prevention of activation-induced T cell anergy in immunotherapeutical trials (28). These are reasons that argue for our bispecific molecule, BiUII, that causes the simultaneous activation of both T cells and accessory cells.
Clinical trials with the 17-1A mAb demonstrated that CDC (complement
dependent cytotoxicity) and ADCC most likely contribute to the observed
antitumor effect. However, the most important class of effector cells,
T lymphocytes, is not activated by this Ab (16). This may
be a drawback because our in vitro assays with a comparable
anti-Ep-CAM mAb revealed reduced activity in terms of tumor cell
killing when compared with BiUII (Fig. 2, ). These results are
consistent with investigations in different mouse tumor models
demonstrating the benefit of the redirection principle by bsAb (Fig. 9) as compared with parental mAbs in vivo
(29, 30, 31, 32).
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).
Although the parental anti-Ep-CAM mAb is able to induce an
antitumor ADCC (data not shown), an elevated cytokine production is not
observed. The same holds true for the IL-2 production by T cells, which
was only slightly increased after addition of the anti-CD3 mAb
26II6 in our experimental setting. Also, the BiUII-mediated production
of IL-2 depends on the presence of the target Ag Ep-CAM. Therefore, our
in vitro assays demonstrate that the interaction of T cells and
accessory cells alone in the absence of Ep-CAM-positive target cells is
not sufficient for the induction of IL-2 (Fig. 7), a fact that reduces
the risk of intolerable systemic IL-2 concentrations during in vivo
application of the bsAb. However, in strong contrast to other reported
bsAb, BiUII activates T cells and accessory cells without the addition
of exogenous IL-2, a fact that is particularly important for in vivo Ab
application. In contrast, Klein et al. (8) demonstrated
recently that exogeneous IL-2 is necessary for a comparable activation
of immune cells in vitro with the intact bsAb, SHR-1. Because SHR-1 is
composed of the subclasses mIgG1 x rat IgG2b, these data argue
for the necessity of two potent subclasses (like mIgG2a x rat
IgG2b) to obtain optimal effector function leading to activation of
accessory cells. Thus, the Tri-cell-complex represents a
self-supporting system that produces cytokines that are necessary for
efficient immune cell activation.
Thus, we postulate that the BiUII-mediated activation of accessory
cells via its Fc region in addition to the recruitment of T cells
contributes in two different ways to tumor cell destruction: 1) The
contribution of accessory cells in the absence of T cells was
demonstrated by cytotoxicity assays using the adherent fraction of
PBMCs (Fig. 3). These data indicate that direct lytic killing
mechanisms like apoptosis and phagocytosis or generally spoken, ADCC,
exerted by different classes of accessory cells, improve tumor
elimination. This holds true especially at decreasing E:T ratios as
shown in Fig. 4, where a CD2+ subset was compared
with whole PBMC. 2) The activation of accessory cells is demonstrated
by the production of cytokines like DC-CK1, IL-1, IL-6, and IL-12.
Our results provide evidence that the simultaneous activation of
different effector cells clearly multiplies the antitumor efficiency
(Figs. 2, 5, 6, and 8). Hence, we postulate that the formation of a
Tri-cell-complex consisting of T cells, accessory cells, and tumor
cells is required for optimal antitumor efficiency (Fig. 9), because a
single class of effector cells or a combination of the two parental Abs
(Fig. 2) was much less efficient.
bsAb are either used as intact molecules or as F(ab')2 fragments. Whereas the antitumor activity is significantly higher with intact bsAb, F(ab')2 fragments are considered to provoke less serious side effects in vivo (33). An excessive production of cytokines is often regarded as the major drawback and limiting factor for in vivo application of intact bsAb. However, in our mouse model system, a comparable bsAb (anti- MHC class II/anti-CD3) with the same Ab isotypes (mouse IgG2a and rat IgG2b) was well tolerated even at concentrations much higher than those which are considered to have therapeutic effects in humans (31).
Another strong argument supporting the use of intact bsAb is the
observed up-regulation of cytokines and activation of accessory cells,
especially DC (Fig. 8), which are prerequisites for the induction of an
antitumor immunity. Anti-Id network responses, as well as cellular and
humoral immune responses, were observed in single cases even after
conventional immunotherapeutical approaches with mAbs. The
Tri-cell-complex seems to be an ideal environment for cell-cell
interactions, allowing the exchange of costimulatory signals between T
cells and accessory cells that amplifies immune reactions. In addition,
we have demonstrated recently that an intact bsAb elevates a
long-lasting tumor immunity in a syngeneic lymphoma mouse model
(34). However, due to the xenogenic origin of BiUII, human
anti-mouse Ab or human anti-rat Ab reactions cannot be excluded,
especially after repeated applications. Therefore, the future aim will
be to evaluate efficacy, side effects, and optimal application modes of
intact bsAb to induce antitumor immunity in animal models and clinical
trials.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Horst Lindhofer, Clinical Cooperation Group "Bispecific Antibodies," Department of Otorhinolaryngology, Ludwig-Maximilians-University, Marchioninistrasse 25, 81377 Munich, Germany. E-mail address:
3 Abbreviations used in this paper: bsAb, bispecific Abs; SCCHN, squamous carcinoma cell lines of the head and neck; DC, dendritic cells; ADCC, Ab-dependent cellular cytotoxicity; Ep-CAM, epithelial cell adhesion molecule.
Received for publication February 8, 1999. Accepted for publication May 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII. J. Hematother. 4:453.[Medline]
RI (CD89) as a novel trigger molecule for bispecific antibody therapy. Blood 90:4485. receptors with rat IgG2b: a new indicator for the FcRIIa (R-H131) polymorphism. J. Immunol. 154:1852.[Abstract]
receptor-mediated phagocytosis by human blood dendritic cells. J. Immunol. 157:541.[Abstract]
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