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NEOPLASIA
From the Clinical Cooperation Group Bispecific
Antibodies of the Department of Otorhinolaryngology, Ludwig Maximilians
University, Munich, Germany; and GSF Institute of Clinical Molecular
Biology and Tumor Genetics, National Research Center for Environment
and Health, Munich, Germany.
Bispecific antibodies (bsAbs) can efficiently mediate tumor cell
killing by redirecting preactivated or costimulated T cells to
disseminated tumor cells, especially in a minimal residual disease
situation. This study demonstrates that the trifunctional bsAb BiLu is
able to kill tumor cells very efficiently without any additional
costimulation of effector cells in vitro and in vivo. Remarkably, this
bsAb also induces a long-lasting protective immunity against the
targeted syngeneic mouse tumors (B16 melanoma and A20 B-cell lymphoma,
respectively). A strong correlation was observed between the induction
of a humoral immune response with tumor-reactive antibodies and the
survival of mice. This humoral response was at least in part tumor
specific as shown in the A20 model by the detection of induced
anti-idiotype antibodies. Both the survival of mice and antitumor
titers were significantly diminished when F(ab')2 fragments
of the same bsAb were applied, demonstrating the importance of the Fc
region in this process. With the use of T-cell depletion, a
contribution of a cellular antitumor response could be demonstrated.
These results reveal the necessity of the Fc region of the bsAb with
its potent immunoglobulin subclass combination mouse immunoglobulin G2a
(IgG2a) and rat IgG2b. The antigen-presenting system seems to be
crucial for achieving an efficient tumor cell killing and induction of
long-lasting antitumor immunity. Hereby, the recruitment and activation
of accessory cells by the intact bsAb is essential.
(Blood. 2001;98:2526-2534) Bispecific antibodies (bsAbs) are regarded as
powerful tools for the treatment of malignant cells in a minimal
residual disease situation, as single disseminated tumor cells are most
easily targeted by an immunologic attack. However, the bsAbs described to date normally activate only a single class of effector cell, ie,
either T cells,1 natural killer cells,2
Fc The successful induction of antitumor immunity was observed in various
murine tumor models by using gene therapy,7 vaccination strategies,8,9 or antibody-mediated
immunotherapy,10,11 demonstrating the feasibility of new
concepts in cancer therapy. However, the transfer of these encouraging
approaches into the clinic is still hampered by certain disadvantages.
One obstacle is the insufficient gene transfer in gene therapy
approaches. Moreover, recent studies elucidate the complexity of T-cell
regulation, revealing that, in addition to CD28, other costimulatory
molecules and cytokines are necessary for appropriate T-cell
activation. In this context, Renner et al12 demonstrated
the need for adhesion molecules such as LFA-1 and CD2 as costimulatory
signaling molecules, rather than as pure cellular contact mediators in
CD3 and CD28 bsAb-stimulated T lymphocytes. Therefore, the gene
transfer of single costimulatory molecules or cytokines into tumor
cells will probably not be sufficient to achieve physiologic T-cell
activation at the tumor site and induction of tumor-specific memory T
cells. This handicap may be overcome by fusing tumor cells with
dendritic cells (DCs)8,13 or by transferring genes
encoding tumor antigens into DCs; however, both approaches are
technically complex and therefore may be unsuitable for routine
clinical application.
In our study, we tried to mimic the natural situation by using intact
bsAbs to redirect not only T cells but also accessory cells to the
tumor site, allowing a simultaneous activation of the
antigen-presenting system. In contrast to other approaches, this
process could be accomplished without the need for complicated gene
transfer or cell fusion techniques. The trifunctional bsAb binds and
activates the T cell by the CD3 molecule so that the activation signal
1 is delivered to the T cell. The activation of accessory cells is
initiated by the binding to the Fc region of the bsAb by Fc Mice and cell lines
Production and purification of bsAb BiLu and
bsF(ab')2
In vitro cytotoxicity assay BsAb-mediated tumor cell killing was measured by a colorimetric MTT-based assay as described previously.19 Briefly, B16 tumor cells transfected with the human EpCAM gene or wild-type B16 tumor cells were coincubated with spleen cells of naive C57BL/6 mice for 24 to 48 hours in 96-well flat-bottom plates at the indicated ratios and in the presence of 50 ng/mL bsAb. Alternatively the antibody concentration was titrated from 50 to 0.05 ng/mL at a fixed E/T ratio of 20:1. To increase T-lymphocyte frequency, spleen cells were reduced of B lymphocytes by panning with anti-IgG+M (Dianova, Hamburg, Germany). As measured by flow-activated cell sorter (FACS) the effector cell population consisted of about 50% CD4+ T cells, 35% CD8+ T cells, 5% macrophages, and 10% remaining B cells (not shown). Then, after removal of effector cells by washing, viable adherent B16 cells were stained with MTT solution (0.5 mg/mL; Sigma, Deisenhofen, Germany) for 4 hours. The MTT solution was removed, and blue crystals of formazan were dissolved in dimethyl sulfoxide. Absorbance was measured with a spectrophotometer at 540 nm. Results were calculated as follows: the percentage of cell death = 100 × (C E)/(C B), where C is the optical density reading
of target cells without effectors (control), B is the 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. In all cases at least triplicates were performed, and
SD was less than 15%.
FACS analysis Target cells (2-4 × 105) were incubated with the primary antibody for 30 minutes on ice in FACS buffer (phosphate-buffered saline with 5% FCS and 0.1% NaN3). After washing, cells were stained with a second fluorescein isothiocyanate (FITC)- or phycoerythrin-labeled antibody, washed, and suspended in FACS buffer with propidium iodide. Flow cytometry was performed by using a FACSCalibur cytometer and the CellQuest analysis program (Becton Dickinson, Heidelberg, Germany). For detection of bsAbs binding on murine CD3, EL-4 lymphoma or spleen cells from mice were used as targets. Binding to human EpCAM was assessed with the transfected cell lines A20-EpCAM, B16-EpCAM, 293Ep, or with human HCT-8 cells. Polyclonal antimouse IgG, antirat IgG (Dianova, Hamburg, Germany), or monoclonal anti-rat IgG2b (ATCC/Tib174) and antimouse IgG2a (R19-15, Pharmingen, Hamburg, Germany) were used as secondary detection antibodies.Assessment for tumor-reactive antibodies The presence of tumor-reactive antibodies in mice sera was determined by flow cytometry. For this purpose mice sera were diluted 1:30 in FACS buffer and incubated with 2 to 4 × 105 B16 or A20 target cells. After washing antibodies bound to tumor cells were detected either by FITC-conjugated polyclonal rat anti-mouse IgG or with IgG subclass-specific antibodies against mouse IgG1 and IgG2a (Pharmingen, Hamburg, Germany). Reactivity was calculated as the percentage of positively stained tumor cells. The nonparametric Mann-Whitney U test was used to evaluate statistical differences between serologic reactions.Detection of EpCAM-specific antibodies Preimmune and postimmune sera of mice were pooled in groups and titrated against human EpCAM-transfected 293Ep cells. Cell-bound antibodies were detected by flow cytometry using a FITC-conjugated goat antimouse IgG1 antibody (Southern Biotechnology Associates, Birmingham, AL). EpCAM specificity of detected antibodies was verified by serum titration against the 293 cell line that does not express the EpCAM
antigen (vector control).
Anti-idiotypic ELISA Antibodies against the A20 Id were measured as follows: ELISA plates were coated with A20 IgG2a purified from culture supernatants, incubated with serially diluted preimmune or immune sera, followed by biotin-labeled goat antimouse IgG1 (Amersham Life Science, Buckinghamshire, United Kingdom), and developed with avidin-peroxidase. Reactivity of the sera with the constant domains of the A20 immunoglobulin was excluded by a similar ELISA using the irrelevant BALB/c-derived IgG2a monoclonal antibody TPA02 (TRION Pharma GmbH, Munich, Germany) as the capturing antigen. Quantification of the anti-Id levels was performed by using the 6D4 (H.L., unpublished data, July 1993) mIgG1 anti-Id antibody against the mIgG2a 7D620 antibody as a standard.In vivo therapy with bsAb C57BL/6 mice received 5 × 103 B16-EpCAM melanoma cells intraperitoneally on day 0. BsAb treatment was started with 2.5 µg BiLu on day 2 and continued with 1 µg each on days 4 and 7. Alternatively, 10 µg bsAb was given on day 2 and another 5 µg on days 4 and 7. The parental group was treated with a combination of the 2 monospecific antibodies C215 and 17A2 by using equivalent doses for each antibody. Control groups received no antibody. Surviving mice were rechallenged with a reduced but still lethal amount of 750 B16-EpCAM cells. Mice were killed after apparent intraperitoneal tumor growth (abdominal swelling) that was confirmed by postmortem dissection. BALB/c mice were challenged with 2 × 106 A20-EpCAM cells intravenously followed by intraperitoneal injection of 4 µg bsAb BiLu or bsF(ab')2 3 hours later. Again the control with parental antibodies was performed. In all cases at least 2 independent experiments were carried out with a minimum of 6 mice per group. Statistical analysis of survival data was performed by using the log-rank test.Vaccinations and adoptive serum transfer Female BALB/c mice 10 to 12 weeks old were immunized against the syngeneic A20 tumor line by intraperitoneal injection of 5 × 104 irradiated (50 Gy) A20-EpCAM or A20 wild-type cells 2 hours after intraperitoneal donation of 4 µg bsAb BiLu or bsF(ab')2 (priming immunization). Four weeks later mice received an identical booster immunization. To evaluate the role of CD4+ T-helper (TH) cells in the immunization process a group of mice was depleted of CD4+ T cells by injection of 400 to 750 µg CD4.2 antibody21 every 3 days before priming and booster immunization. CD4+ or CD8+ T-cell depletion in the effector phase was performed with 500 µg CD4.2 or CD8.2 antibody21 4 days before the tumor challenge. For control, mice were treated only with irradiated tumor cells without bsAb. The final challenge consisted of 7 × 105 viable, untransfected wild-type A20 cells and was performed (intraperitoneally) 6 weeks after the priming immunization. Alternatively, 4 × 105 A20 cells were given intravenously. Blood samples were collected from the tail vein before any treatment (control) and 2 days before the challenge. For adoptive transfer experiments sera of bsAb-immunized mice were pooled, and 300 µL serum was injected intravenously together with 3 × 105 A20 wild-type cells into naive BALB/c mice. Control mice received serum of naive nonimmunized animals in combination with tumor cells. Each experiment has been repeated at least once. All animal groups comprised 6 mice. Statistical analysis of survival curves was performed by using the log-rank test.
Intact bsAb BiLu reveals high antitumor efficacy in vitro and in vivo To evaluate the efficacy of the intact bsAb, BiLu, with specificities anti-CD3 × antihuman EpCAM in redirected lysis of tumor cells, cytotoxicity experiments were carried out in vitro. Spleen cells of naive C57BL/6 mice with an increased T-cell frequency of about 85% were obtained by removing B cells by anti-IgG+M panning. Different than described by others,22,23 thus obtained T cells were not preactivated by interleukin 2 (IL-2) or other stimulatory molecules. Nevertheless, the melanoma line B16-EpCAM was efficiently killed by these effector cells in the presence of bsAb BiLu (Figure 1A). Furthermore, bsAb-mediated lysis was much greater than that achieved by using the parental antibodies at equimolar amounts and was observed over a wide range of bsAb concentrations. Activity was still detected at 5 ng/mL (Figure 1B). Also bsF(ab')2 fragments efficiently induced tumor cell killing, indicating comparable biological activity (Figure 1A,B). Finally, the cell-mediated lysis was mainly antigen specific because B16 wild-type cells that do not express the target antigen EpCAM were only weakly killed (Figure 1A). However, there was a tumor growth inhibition of B16 cells especially at higher E/T ratios. Although we do not know the exact mechanism of this inhibition, bystander effects such as the release of cytokines like tumor necrosis factor or
interferon  might be responsible for this
observation.24 In summary, the bsAb BiLu revealed high and
predominant antigen-specific lytic capacity for syngeneic tumor cells
in vitro.
As a next step, we analyzed the antitumor efficiency of the bsAb
BiLu in vivo. In a therapeutic approach we injected C57BL/6 mice with a
lethal dose of B16-EpCAM melanoma cells intraperitoneally and initiated
bsAb therapy 2 days later. A total dose of 4.5 µg (2.5/1/1 [2.5 µg
on day 2, 1 µg on day 4, and 1 µg on day 7 after tumor
challenge]) was sufficient to cure 100% of the animals, whereas all control mice died within 28 days (Figure
2A). In addition, the therapeutic outcome
of the parental group, which received 4.5 µg of each parental
antibody, was significantly worse (P < .006) as compared
with the bsAb group. In contrast to the observed tumor growth
inhibition of untransfected B16 cells in vitro such an effect was not
seen in vivo: the growth of untransfected B16 wild-type tumor could not
be inhibited or delayed by the bsAb (Figure 2A). This finding clearly
demonstrates the high specificity of bsAb BiLu-induced tumor cell
killing and underlines the importance of retargeted cytotoxicity. To
further evaluate whether these results would be similar with other
tumors, we repeated this experiment in a B-cell lymphoma model with
EpCAM-transfected A20 cells. A single dose of 4 µg bsAb BiLu was
sufficient to inhibit tumor growth with 100% survivors, whereas an
equimolar amount of both parental antibodies led to a significantly
worse inhibition of tumor growth, with 29% survivors
(P = .0009; Figure 2B). These results clearly demonstrated
the benefit of the redirection principle by this bsAb in the B16
melanoma and A20 lymphoma models. Hence, our data are in good
accordance with the findings of other groups that observed the same
superiority of bsAb compared with parental, monospecific antibodies in
different lymphoma models.25-28
BsAb-treated mice develop tumor-reactive antibodies and long-lasting antitumor protection In a second therapy experiment, 14 of 18 bsAb-treated mice survived the primary B16-EpCAM tumor challenge (Figure 3). To analyze differences in immune responses between mice that succumbed to the tumor and those that successfully rejected it, we assessed the sera for tumor-reactive antibodies. Indeed, we found a strong humoral response specific for the tumor in all surviving animals. In contrast, sera of mice that did not survive displayed only a weak reaction (Table 1, mice 15-18). Analysis of the immunoglobulin subclass composition revealed a dominant IgG2a response, whereas no IgG1 antitumor antibodies could be detected. To determine whether antitumor protection was also generated in the surviving animals, we challenged the mice a second time with a lethal number of tumor cells in the absence of bsAb. All animals survived the tumor rechallenge (Figure 3). Consequently, the initial treatment of the tumor with this bsAb led not only to total tumor eradication but also to the induction of immune protection. Notably, all the mice rechallenged on day 144 after the primary challenge were still able to reject the tumor, indicating the high efficacy and long duration of the antitumor response.
BsAb BiLu induces antitumor immunity in the A20 lymphoma model Next, we investigated the exact contribution of humoral and cellular immune responses to the observed antitumor immunization. Having established that the combination of BiLu and vital tumor cells induce an antitumor response, it was important to adapt the immunization protocol to the clinical situation of tumor patients. Therefore, we designed a vaccination strategy with a defined dose of irradiated, proliferation-incompetent tumor cells. We also switched from the aggressively growing B16 melanoma model to the more moderate proliferating A20 lymphoma model. In these experiments BALB/c mice were immunized with irradiated A20-EpCAM cells and bsAb BiLu. Finally, we challenged the mice deliberately with untransfected A20 wild-type cells to see whether an immune response against the whole tumor cell independent of the target antigen EpCAM could be achieved (Figure 4). Again, prior to challenge tumor-reactive antibodies were detectable in the sera of BiLu-treated mice but not in control mice treated only with irradiated tumor cells (Table 2). These data indicated that the intact bsAb was essential for the generation of the humoral antitumor response. Moreover, the presence of induced antibodies against the tumor correlated with the survival of mice. All control mice died, whereas animals treated with intact bsAb and developing tumor-reactive antibodies survived the tumor challenge (Figure 5).
We further analyzed the mice sera for EpCAM-specific antibodies.
Interestingly, the human antigen was not immunogenic per se. Only after
injection of intact bsAb BiLu and A20-EpCAM cells, antibodies against
human EpCAM were generated (Table 3).
Thereby, the induction of an idiotypic network response can be
excluded, because the donation of BiLu in combination with
EpCAM
To determine whether this humoral antitumor response contributed to the
observed protection against the A20 wild-type challenge, we performed
adoptive transfer experiments. Sera of bsAb-immunized mice were pooled
and transferred together with vital A20 cells into naive BALB/c mice.
Although the protection effect was moderate, tumor growth in these mice
was delayed significantly when compared with control mice that received
A20 cells in sera of unimmunized animals (P = .018). This
demonstrated that the obtained antitumor protection was
mediated at least in part by the humoral reaction against the tumor
(Figure 8).
Because the production of antibodies against tumor-specific or
-associated antigens by B lymphocytes requires the support of
CD4+ TH cells, we expected to suppress this
reaction by the application of a depleting anti-CD4 antibody during the
immunization phase. In fact, the generation of antibodies against A20
cells, in general, as well as specific anti-idiotypic antibodies was
diminished after depletion of TH cells (Table 2, Figure 6),
resulting also in a significantly reduced number of surviving animals
(Figure 5 and Figure 9;
P = .02). Therefore, TH cells were mandatory
for bsAb-based induction of tumor-reactive antibodies as well as for tumor protection. Next, we evaluated the role of T cells in the effector phase. To this end, the depletion of CD8+ T cells
caused a decrease of survival rate from 100% to 66% (Figure 9),
whereas the depletion of CD4+ T cells had no effect (not
shown). This finding indicated the participation of cytotoxic
CD8+ T lymphocytes in the eradication of the tumor. Taken
together, these results demonstrated the generation of humoral as well
as cellular immunity against the A20 lymphoma induced in the presence of intact bsAb BiLu.
Fc region of the bsAb BiLu is obligatory for the induction of anti-A20 immunity and efficient tumor cell killing To clarify the role of the Fc region of the bsAb for the induction of antitumor immunity, we evaluated the efficacy of F(ab')2 fragments of BiLu. Complete bsAb was digested with pepsin under limiting conditions and purified on FPLC ion exchange chromatography. FACS analysis and SDS gel electrophoresis proved that biologically active bsF(ab')2 fragments of high purity were obtained (not shown). This finding was confirmed by in vitro cytotoxicity assays in which the mediated tumor cell killing proved to be comparable to intact bsAb (Figure 1). However, these bsF(ab')2 fragments failed to induce an immune protection against A20 lymphoma cells in vivo. Neither tumor-reactive, EpCAM-specific, nor anti-A20 Id antibodies were detectable nor was fast performance liquid chromatography effective protection against the tumor challenge observed (Tables 2 and 3, Figures 5 and 6). These data underline the essential role of the Fc part for the development of protective immunity.Weiner et al29 compared genetically constructed bsF(ab')2 with intact bsAb redirecting CD3 T cells to the 38C13 lymphoma Id. They reported increased T-cell activation and significantly improved therapeutic outcome when complete bsAb was used. In this context, we were also interested in evaluating the direct tumor killing capacity of bsF(ab')2 compared with the intact bsAb BiLu in a therapeutic setting. Therefore, we challenged BALB/c mice with A20-EpCAM cells and began with antibody treatment (4 µg) 3 hours later. Whereas the application of bsAb BiLu resulted in a 100% protection against the tumor, all mice in the bsF(ab')2 group succumbed to the lymphoma (Figure 2B; P < .0001). Even the control group receiving the mixture of parental antibodies had a better outcome in long-term survival. In summary, the Fc portion of the bsAb was of indispensable importance for the therapeutic efficacy as well as for the immunization potency in the A20 tumor model.
In this study, we have used newly designed intact
bsAbs6 that redirect not only T cells but also Fc
receptor+ cells to the tumor site. On the basis of previous
investigations,5 we argue that 2 mechanisms are mainly
responsible for the high antitumor efficacy observed with this reagent.
First, in addition to the recruited T cells, accessory cells are
activated by an interaction between the Fc region of the intact bsAb
and Fc
Obviously, the most interesting feature of this new intact bsAb format is its ability to induce a long-lasting tumor immunity as demonstrated by using 2 different syngeneic mouse tumor models. In the B16 melanoma model even 144 days after the first tumor contact and treatment with bsAb, a second challenge, without bsAb, was rejected in all mice, thereby demonstrating the long-lasting protection mechanism (Figure 3). In contrast, the combinatorial usage of 2 bsF(ab')2 fragments (CD3 × EpCAM + CD28 × EpCAM) in the identical tumor model only led to a marginal tumor elimination and poor induction of tumor immunity.31 This result further supports the hypothesis that the Fc portion of the bsAb is crucial for the killing capacity as well as for the induction of tumor immunity and suggests that single costimulatory signals via CD28 to the T cell are not sufficient to replace physiologic T-cell activation mediated by accessory cells. To adapt the immunization protocol to the clinical situation, we designed a vaccination strategy with a defined dose of irradiated, proliferation-incompetent tumor cells (Figure 4). We also switched from the aggressively growing B16 melanoma model to the more moderate proliferating A20 lymphoma that is more similar to most courses of human cancer. However, in both tumor models a strong correlation was found between the generation of tumor-reactive antibodies after initial bsAb treatment and the survival of mice after rechallenge with tumor cells only. A closer look at the induced humoral response revealed the presence of EpCAM-specific antibodies. The development of such antibodies was strictly restricted to mice vaccinated with intact bsAbs and was independent of an idiotypic network response (Table 3). To clarify that the immune protection was not based only on the artificially introduced human EpCAM antigen, we challenged the mice deliberately with untransfected A20 wild-type cells. In spite of this critical modification, all immunized mice survived as shown in Figure 5. Moreover, in the A20 B-cell lymphoma model we were able to raise an anti-Id response without targeting the tumor-specific Id. These results provide clear evidence that our vaccination strategy yielded immune responses against various antigens of the individual tumor, independent of the surrogate target antigen EpCAM recognized by the bsAb on the tumor cell. The significant loss of immune protection using bsF(ab')2 fragments of the identical bsAb underlines the importance of the Fc region in this process (Figure 5). Importantly, this finding was recognized prior to the wild-type challenge by the failure to detect tumor-reactive antibodies. Therefore, assessment of antitumor antibodies served as a prognostic factor for evaluating the success of the vaccination strategy (Table 2 and Figure 6). By using bsAb and A20 wild-type cells without the target antigen EpCAM, we detected a humoral anti-A20 Id response, although the bsAb could not interact with A20 wild-type cells directly (Figure 6). This immune response is likely due to nonspecific activation of T cells and antigen-presenting cells induced by the trifunctional bsAb. However, the observed antibody response raised by this unspecific activation was rather weak. Moreover, challenging these mice revealed only a partial protection against the tumor (Figure 7). Therefore, optimal immunization and full antitumor protection was only accomplished when trifunctional bsAb and target antigen-expressing cells were combined. Depletion experiments were used to evaluate the role of T cells in both the induction and the effector phase. As shown in Table 2, the induction of the tumor-specific humoral response was clearly diminished (P < .0023) after CD4+ T-cell depletion in the priming phase. Moreover, a significant decrease of survival rate (P = .02) was observed in this group (Figure 9). These results indicated a clear contribution of a cellular immune response to the observed antitumor immunity. This finding could be further confirmed by the depletion of CD8+ T cells in the effector phase, which caused a markedly loss of tumor protection. However, adoptive serum transfer experiments revealed only a weak protection by the isolated use of tumor-reactive antibodies (Figure 8). Taken together, humoral as well as cellular immune responses were generated against A20 lymphoma cells, whereby cell-mediated immunity may be of major importance. Bendandi et al32 recently described an anti-Id vaccination approach for follicular lymphoma in an adjuvant situation, resulting in molecular remissions. However, the encouraging results were hampered by the need to generate individual Id-producing hybridomas. Here, the use of intact bsAb in combination with sorted irradiated tumor cells could allow a simple application that is also capable of inducing an anti-Id response. Although we see no signs of toxicity in the preclinical mouse models, it will be essential to investigate possible side effects and toxicity of this potent immune activation in a clinical setting. In summary, the vaccination strategy described here achieves a long-lasting antitumor immunity without the complexity of gene transfer or cell-fusion techniques and therefore opens new perspectives for a broader clinical application.
We thank D. J. Schendel, M. Roskrow, E. Noessner, R. Zeidler, and R. Mocikat for suggestions and critically reading of the manuscript and P. Reitmeir for helping us with statistical analysis. Expert technical assistance from S. Erndl, S. Wosch, U. Bamberg, and J. Jasny is gratefully acknowledged.
Submitted September 11, 2000; accepted June 12, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Horst Lindhofer, GSF Institute of Clinical Molecular Biology and Tumor Genetics, Marchioninistr.25, 81377 Munich, Germany; e-mail: lindhofer{at}gsf.de.
1. Fanger MW, Segal DM, Wunderlich JR. Going both ways: bispecific antibodies and targeted cellular cytotoxicity [news]. FASEB J. 1990;4:2846-2849[Medline] [Order article via Infotrieve]. 2. Weiner LM, Clark JI, Ring DB, Alpaugh RK. Clinical development of 2B1, a bispecific murine monoclonal antibody targeting c-erbB-2 and Fc gamma RIII. J Hematother. 1995;4:453-456[Medline] [Order article via Infotrieve]. 3. Heijnen IA, Rijks LJ, Schiel A, et al. Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies. J Immunol. 1997;159:5629-5639[Abstract].
4.
Valerius T, Stockmeyer B, van Spriel AB, et al.
FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy.
Blood.
1997;90:4485-4492
5.
Zeidler R, Reisbach G, Wollenberg B, et al.
Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing.
J Immunol.
1999;163:1246-1252 6. Lindhofer H, Mocikat R, Steipe B, Thierfelder S. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies. J Immunol. 1995;155:219-225[Abstract].
7.
Dranoff G, Jaffee E, Lazenby A, et al.
Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.
Proc Natl Acad Sci U S A.
1993;90:3539-3543 8. Gong J, Chen D, Kashiwaba M, Kufe D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat Med. 1997;3:558-561[CrossRef][Medline] [Order article via Infotrieve].
9.
Mocikat R, Selmayr M, Thierfelder S, Lindhofer H.
Trioma-based vaccination against B-cell lymphoma confers long-lasting tumor immunity.
Cancer Res.
1997;57:2346-2349 10. Guo YJ, Che XY, Shen F, et al. Effective tumor vaccines generated by in vitro modification of tumor cells with cytokines and bispecific monoclonal antibodies. Nat Med. 1997;3:451-455[CrossRef][Medline] [Order article via Infotrieve]. 11. French RR, Chan HT, Tutt AL, Glennie MJ. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med. 1999;5:548-553[CrossRef][Medline] [Order article via Infotrieve]. 12. Renner C, Jung W, Sahin U, van Lier R, Pfreundschuh M. The role of lymphocyte subsets and adhesion molecules in T cell-dependent cytotoxicity mediated by CD3 and CD28 bispecific monoclonal antibodies. Eur J Immunol. 1995;25:2027-2033[Medline] [Order article via Infotrieve]. 13. Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med. 2000;6:332-336[CrossRef][Medline] [Order article via Infotrieve]. 14. Zeidler R, Mysliwietz J, Csanady M, et al. The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells. Br J Cancer. 2000;83:261-266[CrossRef][Medline] [Order article via Infotrieve].
15.
Litvinov SV, Velders MP, Bakker HA, Fleuren GJ, Warnaar SO.
Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule.
J Cell Biol.
1994;125:437-446
16.
Dohlsten M, Hansson J, Ohlsson L, Litton M, Kalland T.
Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.
Proc Natl Acad Sci U S A.
1995;92:9791-9795 17. Miescher GC, Schreyer M, MacDonald HR. Production and characterization of a rat monoclonal antibody against the murine CD3 molecular complex. Immunol Lett. 1989;23:113-118[CrossRef][Medline] [Order article via Infotrieve]. 18. Clark M, Gilliland L, Waldmann H. Hybrid antibodies for therapy. Prog Allergy. 1988;45:31-49[Medline] [Order article via Infotrieve].
19.
Heo DS, Park JG, Hata K, Day R, Herberman RB, Whiteside TL.
Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity.
Cancer Res.
1990;50:3681-3690 20. Coulie PG, Uyttenhove C, Wauters P, et al. Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3. Eur J Immunol. 1991;21:1703-1709[Medline] [Order article via Infotrieve]. 21. Reinecke K, Mysliwietz J, Thierfelder S. Single as well as pairs of synergistic anti-CD4+CD8 antibodies prevent graft-versus-host disease in fully mismatched mice. Transplantation. 1994;57:458-461[Medline] [Order article via Infotrieve]. 22. Hombach A, Mathas S, Jensen M, et al. Activation of resting T cells against the CA 72-4 tumor antigen with an anti-CD3/CA 72-4 bispecific antibody in combination with a costimulatory anti-CD28 antibody. Anticancer Res. 1997;17:2025-2032[Medline] [Order article via Infotrieve].
23.
Daniel PT, Kroidl A, Kopp J, et al.
Immunotherapy of B-cell lymphoma with CD3x19 bispecific antibodies: costimulation via CD28 prevents "Veto" apoptosis of antibody-targeted cytotoxic T cells.
Blood.
1998;92:4750-4757 24. Qian JH, Titus JA, Andrew SM, et al. Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth. J Immunol. 1991;146:3250-3256[Abstract]. 25. Weiner GJ, Hillstrom JR. Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma. J Immunol. 1991;147:4035-4044[Abstract].
26.
Renner C, Jung W, Sahin U, et al.
Cure of xenografted human tumors by bispecific monoclonal antibodies and human T cells.
Science.
1994;264:833-835
27.
Bohlen H, Manzke O, Titzer S, et al.
Prevention of Epstein-Barr virus-induced human B-cell lymphoma in severe combined immunodeficient mice treated with CD3xCD19 bispecific antibodies, CD28 monospecific antibodies, and autologous T cells.
Cancer Res.
1997;57:1704-1709
28.
Lindhofer H, Menzel H, Gunther W, Hultner L, Thierfelder S.
Bispecific antibodies target operationally tumor-specific antigens in two leukemia relapse models.
Blood.
1996;88:4651-4658 29. Weiner GJ, Kostelny SA, Hillstrom JR, et al. The role of T cell activation in anti-CD3 x antitumor bispecific antibody therapy. J Immunol. 1994;152:2385-2392[Abstract].
30.
De Jonge J, Heirman C, de Veerman M, et al.
In vivo retargeting of T cell effector function by recombinant bispecific single chain Fv (anti-CD3 x anti-idiotype) induces long-term survival in the murine BCL1 lymphoma model.
J Immunol.
1998;161:1454-1461 31. Grosse-Hovest L, Brandl M, Dohlsten M, Kalland T, Wilmanns W, Jung G. Tumor-growth inhibition with bispecific antibody fragments in a syngeneic mouse melanoma model: the role of targeted T-cell co-stimulation via CD28. Int J Cancer. 1999;80:138-144[CrossRef][Medline] [Order article via Infotrieve]. 32. Bendandi M, Gocke CD, Kobrin CB, et al. Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat Med. 1999;5:1171-1177[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  F. Hirschhaeuser, T. Leidig, B. Rodday, C. Lindemann, and W. Mueller-Klieser Test System for Trifunctional Antibodies in 3D MCTS Culture J Biomol Screen, September 1, 2009; 14(8): 980 - 990. [Abstract] [PDF]
|
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 M. Jager, A. Schoberth, P. Ruf, J. Hess, and H. Lindhofer The Trifunctional Antibody Ertumaxomab Destroys Tumor Cells That Express Low Levels of Human Epidermal Growth Factor Receptor 2 Cancer Res., May 15, 2009; 69(10): 4270 - 4276. [Abstract] [Full Text] [PDF]
|
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|
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|
 P. WIMBERGER, M. HEUBNER, H. LINDHOFER, M. JAGER, R. KIMMIG, and S. KASIMIR-BAUER Influence of Catumaxomab on Tumor Cells in Bone Marrow and Blood in Ovarian Cancer Anticancer Res, May 1, 2009; 29(5): 1787 - 1791. [Abstract] [Full Text] [PDF]
|
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 B. D. Cheson and J. P. Leonard Monoclonal Antibody Therapy for B-Cell Non-Hodgkin's Lymphoma N. Engl. J. Med., August 7, 2008; 359(6): 613 - 626. [Full Text] [PDF]
|
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|
|
 |
|
 A. Burges, P. Wimberger, C. Kumper, V. Gorbounova, H. Sommer, B. Schmalfeldt, J. Pfisterer, M. Lichinitser, A. Makhson, V. Moiseyenko, et al. Effective Relief of Malignant Ascites in Patients with Advanced Ovarian Cancer by a Trifunctional Anti-EpCAM x Anti-CD3 Antibody: A Phase I/II Study Clin. Cancer Res., July 1, 2007; 13(13): 3899 - 3905. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 A. Natsume, M. Wakitani, N. Yamane-Ohnuki, E. Shoji-Hosaka, R. Niwa, K. Uchida, M. Satoh, and K. Shitara Fucose Removal from Complex-Type Oligosaccharide Enhances the Antibody-Dependent Cellular Cytotoxicity of Single-Gene-Encoded Bispecific Antibody Comprising of Two Single-Chain Antibodies Linked to the Antibody Constant Region J. Biochem., September 1, 2006; 140(3): 359 - 368. [Abstract] [Full Text] [PDF]
|
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|
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|
P. Kiewe, S. Hasmuller, S. Kahlert, M. Heinrigs, B. Rack, A. Marme, A. Korfel, M. Jager, H. Lindhofer, H. Sommer, et al. Phase I Trial of the Trifunctional Anti-HER2 x Anti-CD3 Antibody Ertumaxomab in Metastatic Breast Cancer. Clin. Cancer Res., May 15, 2006; 12(10): 3085 - 3091. [Abstract] [Full Text] [PDF]
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 S. Morecki, H. Lindhofer, E. Yacovlev, Y. Gelfand, and S. Slavin Use of trifunctional bispecific antibodies to prevent graft versus host disease induced by allogeneic lymphocytes Blood, February 15, 2006; 107(4): 1564 - 1569. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
mouse immunoglobulin G2a (IgG2a) × rat IgG2b
-mercaptoethanol, 2 mM glutamine, 1 × nonessential amino acids, and 100 U/mL penicillin and streptomycin (complete media). Additionally, Geneticin G418 was
added to B16-EpCAM cells (0.5 mg/mL), and A20-EpCAM cells were grown in
complete media containing Hygromycin B (0.5 mg/mL).
) or bsF(ab')2 (
) targeted against
transfected B16-EpCAM cells. To differentiate antigen-independent cell
lysis by the bsAb, nontransfected B16 target cells were used (bsAb
wild-type, 
). (B) At an E/T ratio of 20:1, the bsAb BiLu (
). B16-EpCAM cells were used as
targets. Effector cell-induced background lysis in the absence of
antibody was subtracted.
) (n = 6) was
treated with a combination of the 2 monospecific antibodies C215
(antihuman EpCAM) and 17A2 (antimurine CD3). The control group (+)
(n = 6) received no antibody. To clarify the antigen dependency of
the treatment one group of mice (
) (n = 8) 3 hours later. Again, a control group with parental antibodies (
A20 wild-type cells did not lead to an immune
response against the tumor antigen. Remarkably, mice developed a
humoral response and were protected against wild-type A20 cells,
although they were immunized with transfected A20-EpCAM cells. As a
consequence, an antitumor response against antigens other than the
target antigen EpCAM must have been induced. An absolutely
tumor-specific antigen of B-cell lymphomas is the immunoglobulin Id. To
investigate whether this antigen was targeted, we also looked for
anti-Id antibodies. Indeed, we found significant titers against the A20
immunoglobulin Id after we immunized mice with bsAb BiLu and A20-EpCAM
cells according to our vaccination protocol (Figure
, CD4-I). Control mice (+) were not pretreated. All mice received
an intravenous tumor challenge of 4 × 105 A20 cells. 