Cancer Research 2010 AACR Elections Sign up for Cancer Research eTOC's
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 1961-1965, April 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bauer, S.
Right arrow Articles by Pfreundschuh, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bauer, S.
Right arrow Articles by Pfreundschuh, M.
[Cancer Research 59, 1961-1965, April 15, 1999]
© 1999 American Association for Cancer Research

Immunology

Immunotherapy of Human Tumors with T-Cell-activating Bispecific Antibodies

Stimulation of Cytotoxic Pathways in Vivo1

Stefan Bauer, Christoph Renner2, Jan-Peter Juwana, Gerhard Held, Sascha Ohnesorge, Klaus Gerlach and Michael Pfreundschuh

Medical Department I, Saarland University Medical School, D-66421 Homburg, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bispecific monoclonal antibodies (Bi-mAbs) specific for a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes represent one of the most successful experimental strategies for the immunotherapy of cancer. We report that the in vivo administration of both {alpha}-CD3/CD30 and {alpha}-CD28/CD30 Bi-mAbs results in the specific activation of xenotransplanted, resting human T cells infiltrating the CD30-positive Hodgkin’s tumor. Bi-mAb treatment resulted in enhanced expression of cytokines such as interleukin 1ß, interleukin 2, tumor necrosis factor type {alpha}, and activation markers including Ki-67, CD25, and CD45RO in tumor-infiltrating lymphocytes. This antigen-dependent, local T-cell stimulation led to the activation of the cytolytic machinery in T lymphocytes, determined by the up-regulation of mRNA-encoding perforin and the cytotoxic serine-esterases granzymes A and B. The Bi-mAb-induced generation of CTLs depended on the presence of the CD30 antigen and the combined application of both Bi-mAbs. Our findings suggest that the combined application of T-cell-activating Bi-mAbs is able to achieve a tumor site-specific activation of the T-cell cytolytic machinery in vivo. The fact that these cytotoxic cells do not home in tumor-associated antigen-negative tissue and do not enter circulation might explain our previous observations (C. Renner et al., Blood, 87: 2930–2937, 1996) of a high cure rate in preclinical models even at an advanced stage of disease.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cells are commonly recognized as the most potent effector cells to generate antitumor cytotoxicity (1) . Many approaches of experimental cancer treatment try, therefore, to induce T-cell immunological effector mechanisms leading to tumor eradication (2 , 3) . One of the most successful strategies is represented by Bi-mAbs,3 which mimic the physiological T-cell stimulation initiated originally by an antigen-presenting cell. Bi-mAbs that target with one arm a TAA and with the other arm a T-cell triggering molecule are able to activate specifically resting T cells, which results in tumor destruction (4 , 5) . According to the current concept of T-cell activation (6) , a combination of two Bi-mAbs that recognize with one site a TAA and with the other the CD3 or CD28 antigen on T cells, respectively, are needed for an antigen-specific-but-MHC-unrestricted T-cell activation (7 , 8) . Using Hodgkin’s lymphoma as a model, we demonstrated that the bridging of Hodgkin’s tumor cells to T-cell triggering molecules by a combined {alpha}-CD30/CD3 and {alpha}-CD30/CD28 Bi-mAb regimen induced an efficient tumor cell lysis in vitro and in vivo (9) . In a preclinical model, the treatment of SCID mice harboring xenografted disseminated CD30+ tumors resulted in the complete cure of all of the animals. Even if the beginning of treatment was delayed and tumor growth advanced, complete remissions could be achieved (10) . Our data confirmed previous observations (11) that only the combination of both CD4+ and CD8+ lymphocytes resulted in the cure of tumor-bearing SCID mice, which suggested that both subsets are crucial for the efficient eradication of tumor cells (10) .

However, the precise way by which Bi-mAb-activated T cells eliminate tumor cells still has to be elucidated. Early experiments with CTLs stressed the role of cytokines in tumor cell lysis (12) . These results were recently challenged by experimental evidence that the contribution of cytokines to tumor cell lysis is not by direct participation in processes involved in rapid cell destruction (13) ; rather, cytokines are now believed to function primarily by down-regulating the cell-cycle activity of tumor cells and by augmenting the cytolytic activity of effector lymphocytes (14) . The key components in the cytolytic machinery of T cells are represented by perforin and granzymes; as potent cytotoxic molecules they can suppress tumor growth (15) . After attaching at the target cells, perforin monomers assemble to polymeric pore structures, which are inserted into the plasma membrane thus causing osmotic cytolysis and the typical picture of necrosis (16) . Secreted granzymes enter the target cell using the perforin-channel and initiate an internal disintegration pathway that leads to DNA fragmentation (apoptosis) and lysis (17) . In vitro data showed that this granule-exocytosis pathway plays a central role in bispecific-antibody-induced T-cell-mediated tumor destruction (18) . In accordance with the observation that CD8+CD45RO+ cells are the most efficient cytotoxic cells, the up-regulation of expression of perforin and granzymes A and B reached the highest levels in this cell population (19) . To investigate whether these molecular programs of Bi-mAb-induced cytotoxic T cells are also operative in the immune destruction of tumor cells in vivo, we established an experimental tumor model in C.B-17 SCID/SCID mice. Simultaneous growth of either CD30-positive Hodgkin’s lymphoma and TAA-unrelated colon carcinoma was induced s.c. Our investigations show that human lymphocytes activated by the combination of both Bi-mAbs are found only in Hodgkin’s lymphoma tissue and to a much lesser extent or not at all in TAA-negative tissue including the unrelated colon carcinoma. This site-specific activation of resting human T lymphocytes led to directed exocytosis of perforin and granzymes, demonstrating that this pathway is a very important mechanism for Bi-mAb-mediated tumor destruction in vivo and the consecutive cure of animals xenografted with disseminated Hodgkin’s lymphoma, even in advanced stages of disease.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Pathogen-free, 4–6-week-old mice with SCID (C.B-17 lcr SCID/SCID) were obtained from the Institut für Versuchstierzucht (Hannover, Germany). Animals were housed and bred in laminar flow racks and fed with autoclaved standard chow and water. "Leaky" animals were identified by ELISA (20) and excluded from further treatment. For in vivo-depletion of murine natural killer cells, all of the mice used in the experiments were pretreated with 100 µl of anti-asialo-GM1-antibody (Wako, Japan) i.p. as described previously (21) . {smhd4}Antibodies and Cell Lines.%The generation, purification, and characterization of the Bi-mAb ({alpha}-CD3/CD30 and {alpha}-CD28/CD30) have been described previously (9 , 22) . The established CD30+ human tumor cell line L540CY has been described previously (23) . The LOVO colon carcinoma-derived line was obtained from American Type Culture Collection. The following moAbs were used for this study: (a) 2H4 (antihuman TNF-{alpha}) and B-G-5 (antihuman IL-2; Boehringer, Mannheim, Germany); (b) ß-36 (antihuman IL-1ß) and goat-antimouse-IgG-FITC (Dianova, Hamburg, Germany); (c) Ki-67-FITC (antihuman Ki-67), ACT1-FITC (antihuman CD25), and UCHL-1 (antihuman CD45RO; Dakopatts, Hamburg, Germany); (d) SK7-FITC and SK7-PE (Phycoerythrin, antihuman CD3); and (e) SK3-PE (antihuman CD4), SK1-PE (antihuman CD8; Becton Dickinson, Heidelberg, Germany).

T-Cell Preparations.
Peripheral blood mononuclear cells from heparinized blood of healthy donors were isolated and T cells were negatively enriched by magnetic activated cell sorting as described previously (22) . Contaminating cell fractions were always less than 0.5%.

Tumor Model.
The experimental model based on the solid growth of two different tumors in the same mice: 2 x 107 L540CY cells (suspended in PBS at a volume of 400 µl) expressing the CD30 Hodgkin’s-associated antigen were applied s.c. in the left ventral thoracic wall. The s.c. injection of the CD30-negative LOVO cell line (1.5 x 107 cells, suspended in PBS at a volume of 400 µl) was given in the right ventrogluteal region of the same animal. Solid tumors were established in 40 SCID mice by this way. Previous experiments had proven a 100% tumor take rate at this cell number. When tumors reached a diameter of 1 cm, bispecific antibodies followed by resting human T lymphocytes were injected into the ventral or dorsal tail vein. There were at least four experimental groups (4–10 mice per group): (a) CD3+ lymphocytes treatment group (1 x 107 T cells, suspended in PBS at a volume of 300 µl); (b) CD3+ lymphocytes and 100 µg of 2F10-Bi-mAbs ({alpha}-CD28/CD30); (c) CD3+ lymphocytes and 100 µg of HT3-Bi-mAbs ({alpha}-CD3/CD30); and (d) CD3+ lymphocytes and 50 µg of each Bi-mAb ({alpha}-CD3/CD30 and {alpha}-CD28/CD30). Seven days after the administration of the Bi-mAbs and effector cells, respectively, the mice were killed by cervical dislocation. The solid tumors, the spleens, and blood were removed for additional investigations.

FACScan Analysis.
Single cell suspensions were obtained by mechanical dissection of different organs. Human lymphocytes (5000) were analyzed in each experiment. For intracellular staining of IL-1ß, IL-2, and TNF-{alpha}, cells were permeabilized by 0.5% Saponin (Sigma, Munich, Germany), and FACS analysis was performed according to the method described previously (24 , 25) . A FITC-conjugated goat-antimouse F(ab') immunoglobulin monomer antibody was used as second-step reagent (Dianova, Hamburg, Germany). For visualizing membrane-bound T-cell activation markers (Ki-67, CD25, CD45RO, CD3, CD4, and CD8) a double-immunofluorescent assay was performed either using two primarily FITC- or PE-conjugated moAbs or using the second-step reagent as described previously (24) .

RNA Isolation.
Portions of removed tissue were snap-frozen in liquid nitrogen and stored at -70°C for RNA extraction. The tissue was homogenized with a tissue tearer. Total RNA was extracted with guanidinium isothiocyanate (26) and quantified by spectrophotometry.

RT-assisted PCR.
RT-PCR was done as described previously (27) . In brief, 2 µg of total RNA in a volume of 4 µl were reverse-transcribed in a 20-µl vol. containing 5 µl of Oligo dT18 primer (10 µM, Life Technologies, Inc., Karlsruhe, Germany), 1 µl Superscript RT (Life Technologies, Inc., Karlsruhe, Germany), 2 µl of Dithiothreitol (Serva, Heidelberg, Germany), and 4 µl of 5-fold-reaction buffer (Life Technologies, Inc.). The reaction mixtures were incubated at 37°C for 1 h, heated at 70°C (10 min), and stored at 4°C. Amplification of specific cDNA-fragments was performed as described by Nakajima et al. (27) . The following primer pairs were used:

(a) the perforin PCR product was generated with 5'-GTCACGTCGAAGTACTTGGTG- 3' and 5'-ATGGCTGATAGCCTGTCTCAG- 3';

(b) granzyme A was generated with 5'-CTGTAACTTGAACAAAAGCT- 3' and 5'-CCAGAATCTCCATTGCACGA- 3';

(c) granzyme B was generated with 5'-GCCCACAACATCAAAGAACAG- 3' and 5'-AACCAGCCACATAGCACACAT- 3'; and

(d) ß-actin was generated with 5'-TCTACAATGAGCTGCGTGTGG- 3' and 5'-CATGAGGTAGTCTGTCAGGTC- 3'.

One µl of cDNA from a total of 20 µl was amplified with 1 unit of Goldstar Taq DNA polymerase (Leiden, Netherlands) in a final volume of 50 µl containing 5 µl of nucleosidtriphosphate (2 mM), 3.3 µl of MgCl2 (Merck, Darmstadt, Germ-any), 1.5 of µl of each primer (10 mM), 5 µl of 10x PCR buffer (Leiden, Netherlands) and Aqua dest. PCR amplification was carried out on a thermocycler (MWG-Biotech, Ebersberg, Germany) with the following profile: 95°C for 80 s, annealing for 80 s, and extension at 72°C for 2 min. The number of cycles and the annealing temperature depended on the primers used: (a) perforin (57°C, 30 cycles); (b) granzyme A (50°C, 32 cycles); (c) granzyme B (61°C, 30 cycles); and (d) ß-actin (61°C, 30 cycles). Aliquots (20 µl) of each PCR product were separated on a 2.5% agarose gel and visualized by ethidium bromide staining (Sigma).

Northern Blot Analysis.
Northern blots were performed as described previously (19) . [32P]CTP-random-labeled (Ready-To-Go Kit, Pharmacia, Freiburg, Germany) gene-specific cDNA probes were hybridized in a formamide buffer system (5 ml of formamide, 2.5 ml of 20x SSC, 1 ml of 50x Denhardt’s solution, 0.5 ml of SDS 20%, 1 ml of double-density H20, and 50 µg/ml heat-denatured salmon sperm) at 42°C for 10–16 h. After hybridization, membranes were washed and exposed at -70°C. The perforin and granzymes A- and B-specific cDNAs were generated by PCR as described previously (18) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bi-mAb-induced Secretion of T-Cell Cytokines and Expression of Activation Markers in Vivo.
CD28-costimulation of TCR/CD3-activated T lymphocytes can augment cell proliferation and production of multiple cytokines (e.g., IL-2 and TNF-{alpha}) and enhance expression of the high-affinity receptor for IL-2 (CD25; Ref. 28 ). As demonstrated by FACScan-analysis (Fig. 1)Citation , the same effect can be achieved by the combined application of our Bi-mAbs, cross-linking the CD3 and CD28 antigen on resting T cells in the presence of CD30+ tumor cells. The highest number of proliferating T cells expressing the cell nucleus-associated antigen Ki-67 as a marker for human cells entering the cell cycle was measured in the CD30-positive Hodgkin’s lymphoma when activated appropriately by both Bi-mAbs (HT3 and 2F10). The majority of stimulated lymphocytes remained in the TAA-positive tissue and did not re-enter into circulation. Neither the TAA-negative tissue nor the unrelated colon carcinoma showed relevant counts of anti-CD3-/anti-Ki-67 double-positive stained cells. CD3+ lymphocytes from those animals treated with one Bi-mAb alone (HT3 or 2F10, respectively) or from the control-group rested in the G0 phase of the cell cycle. Proliferating T cells from the group of mice with combined Bi-mAb treatment showed 2- to 4-fold higher peak levels of IL-1ß, IL-2, and TNF-{alpha} when compared with all of the other treatment-groups. Cross-reactivity of the anti-IL-2-moAb between murine and human IL-2 was excluded by using double immunofluorescence. The high expression level of the CD25 antigen on TILs after combined Bi-mAb treatment confirmed their activation status.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. FACScan-analysis of cytokine-secretion and antigen-expression of xenografted resting human T lymphocytes after in vivo activation by combined application of {alpha}-CD3/CD30 and {alpha}-CD28/CD30 Bi-mAbs. Solid tumor-bearing mice were treated by i.v. injection of resting human T cells followed by Bi-mAb application. Animals were killed after 7 days, and tissues were removed to evaluate the in situ activation of T cells. A, cytokine levels or activation marker expression of anti-CD3-PE-gated T lymphocytes were measured by intra-/extracellular staining with secondary FITC-conjugated moAbs. Columns, the percentage of cytokine/antigen positive cells within the pool of activated CD3+ T cells (mean ± SD; ten mice per treatment-group). Animals were treated with HT3+2F10 ({square}), HT3 ({blacksquare}), 2F10 ( ), or PBS ( ), and expression of indicated cytokines/proliferation markers were measured. The type of tumor and normal tissue analyzed is stated on the X axis. B, differential infiltration of established Hodgkin’s tumors with CD45RO+ memory cells depending on antibody treatment. Simultaneously growing Hodgkin’s and colon tumors were resected from mice treated with different antibody combinations [HT3+2F10 ({square}), HT3 ({blacksquare}), 2F10 ( ), or PBS ( ), and tumor tissue was analyzed for infiltration with CD45RO+ cells. Within the CD45RO+ population, cells were further divided into CD8+ and CD4+ lymphocytes. For each antibody group, values obtained from colon tumors were subtracted from values obtained from Hodgkin’s tumors to correct for unspecific lymphocyte homing in antigen-negative tissue.

 
The in situ activation of the resting human T lymphocytes via cross-linking of both CD3/CD30 and CD28/CD30 Bi-mAbs by solid-growing CD30+-Hodgkin’s lymphoma led to the expansion of the memory lymphocyte pool characterized by the expression of the CD45RO "memory" antigen. Lymphocytes expressing the CD8+CD45RO phenotype are the most potent mediators of target cell lysis in vitro and induce a significant higher rate of cytotoxicity when compared with naive CD8+ lymphocytes characterized by the CD45RA antigen (19) . Lymphocytes infiltrating Hodgkin’s tumors were represented by both CD4+CD3+ and CD8+CD3+ T cells (Fig. 1B)Citation , but the memory antigen was significantly higher expressed by CD8+ cells.

Up-regulation of Perforin and Granzyme Message in Bi-mAb-activated T Cells in Vivo.
As perforin and the granzyme family seem to be the most important mediators involved in target cell lysis, we looked for the up-regulation of these molecules in Bi-mAb-activated tumor infiltrating lymphocytes by Northern blot and RT-PCR analysis. Perforin and granzyme A and B mRNA were expressed only in Hodgkin’s lymphoma of SCID-mice treated with the Bi-mAb combination regimen (Fig. 2Citation , animal 1, Lane 2) and was not detectable by Northern blot in the CD30-negative colon carcinoma (Lane 1) nor in the spleen (Lane 3) or blood (Lane 4). There was no induction of these cytocidal proteins in animals treated with one Bi-mAb alone because detection of granzyme and perforin gene expression failed. Missing of cytotoxic activity after inappropriate treatment is represented by animal 2 (Fig. 2)Citation , which was treated with anti-CD3/CD30 Bi-mAb only. The differential pattern of mRNA expression for perforin and granzymes A and B appeared to be a consequence of the treatment regimen used because the expression of ß-actin gene was similar in all of the tissues resected from different experimental groups.



View larger version (71K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Analysis of perforin and granzymes A and B mRNA expression in different tissues after antibody treatment. Total RNA for Northern blot analysis was isolated from resected tissues as described in "Materials and Methods." Mice were treated with either a combination of both Bi-mAbs (animal 1) or just the HT3 Bi-mAb (animal 2). The following tissues were resected and analyzed: Hodgkin’s tumor (Lane 2); spleen (Lane 3); blood (Lane 4); or an unrelated colon carcinoma (Lane 1). Blots were probed with cDNAs specific for perforin or granzyme A or B. A ß-actin-specific probe was used to ensure equivalent RNA load in all of the lanes.

 
Dependence of Bi-mAb-induced Cytotoxicity on the Expression of the CD30 Antigen in Vivo.
In a second set, we performed a RT-PCR analysis (Fig. 3)Citation to confirm the Northern blot results and to clarify that the Bi-mAb-mediated induction of T-cell cytotoxicity is strictly dependent on the targeted TAA. The different methods revealed adequate results; consistent induction of perforin and granzymes A and B mRNA was dependent on the presence of the CD30 antigen and the combined application of the anti-CD3/CD30 and anti-CD28/CD30 Bi-mAb. The key cytotoxic components were only detectable in the Hodgkin’s tumors of those mice treated with the combined Bi-mAb regimen (Lanes 1–4) and could not be detected in tissues from mice treated with Bi-mAb (Lanes 6-9) or lymphocytes only. The number of cycles used for RT-PCR were established by initial experiments in which varying amounts of activated lymphocytes were mixed with a constant number of tumor cells. The conditions were adjusted to pick up 1 activated lymphocyte within 10.000 tumor cells (data not shown). We could not detect perforin and granzyme message by RT-PCR in our cell lines (data not shown).



View larger version (61K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. RT-PCR analysis of perforin and granzymes A and B expression in different tissues after antibody treatment. RT-PCR for perforin, granzymes A and B, and ß-actin was performed as described in "Materials and Methods." Lane 1–4 represent Hodgkin’s tumors resected from four mice treated with a combination of both Bi-mAbs. DNA marker is shown in Lane 5 for all of the samples. Lane 6–9 represent cDNA generated from different tissues resected from mice treated with a combination of both Bi-mAbs. The order of the tissues analyzed is as follows: spleen (Lane 6); Hodgkin’s lymphoma (Lane 7); peripheral blood (Lane 8); and a colorectal carcinoma as CD30 antigen-negative tumor control (Lane 9).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present report supports our concept that the combination of two Bi-mAbs with reactivity to the CD3 and CD28 antigen on T cells and a TAA on tumor cells, respectively, represents a very effective regimen for the treatment of human tumors. Evaluating the role that different lymphocyte subsets play in tumor cell killing revealed that the results of our in vivo studies were in accordance with the results obtained earlier in vitro (22) . Both CD4+ and CD8+ cells contributed to the Bi-mAb mediated cytotoxicity, with the CD8+ lymphocytes representing the most effective subset (22) . However, the precise way in which Bi-mAb-activated lymphocytes induced tumor cell lysis in vivo still remained uncertain and had not yet been addressed. Several studies dealing with this topic thus far proved that Bi-mAbs and Bi-mAb-coated lymphocytes accumulated at the tumor site and reached significantly higher distribution values in tumor tissue when compared with antigen-negative tissue (9 , 29) . No significant difference in lymphocyte accumulation could be observed in our previous studies between mice treated with one Bi-mAb alone or a combination of both. We favor the hypothesis that the successful eradication of human tumors was not due to quantitative differences in lymphocytes targeted to the tumor site but was rather caused by in situ activation of human T cells entering the tumor compartment. The present SCID mouse model with solid Hodgkin’s and colon tumors established in the same mouse at the same time confirmed this hypothesis inasmuch as expression levels of cytokines that were measured in different tissues revealed a strict dependence on the expression of the CD30 antigen. The concentration of IL-1ß, IL-2, and TNF-{alpha} and the expression values for CD25 and Ki-67 were manifoldly higher in lymphocytes infiltrating Hodgkin’s lymphoma than in colon tumor tissue. Moreover, we showed that the majority of activated T cells remained in the CD30+ tissue and did not enter the circulation. Efficient stimulation of CD3+ lymphocytes was induced only after injection of both CD3/CD30 and CD28/CD30 Bi-mAbs. The crucial role of the second signal for efficient T-cell activation in vivo has been emphasized recently by clinical data. Patients with B-cell lymphoma treated with one anti-CD3/CD19 Bi-mAb alone had only slightly enhanced levels of cytokines with no significant T-cell enrichment in tumor tissue could be observed (30) . However, the addition of IL-2 as second signal initiated efficient T cell activation proven by the increased expression of CD25 and the high levels of cytotoxic granules in tumor infiltrating lymphocytes (31) .

Analyzing the subsets of T cells infiltrating Hodgkin’s lymphoma showed the highest levels for the CD8+CD45RO+ pool. The phenomenon that memory CD8+ T-cells are in vitro much more potent effector cells than naive CD8+ T cells was first described by Azuma et al. for CTL clones (32) and could be confirmed by our group for Bi-mAb-mediated cytotoxicity (22) . Moreover, Demanet et al. (4) demonstrated in an in vivo murine lymphoma model that unprimed mice did not reject tumors efficiently despite the fact that two T-cell activating antibodies were used. However, if animals were primed before treatment with allogeneic EL 4 lymphoma cells, a rapid expansion of T cells expressing the CD45RO memory antigen could be detected leading to a high remission rate after subsequent tumor challenge and treatment with two Bi-mAbs, one recognizing the CD3 antigen and the other one the CD28 antigen on T cells. Therefore, memory T cells seem to have a crucial impact on tumor cell elimination. In our model, the combined application of Bi-mAbs initiating the primary and the costimulatory signal at the tumor site ensured the TAA-dependent expansion of cytotoxic T cells expressing the memory phenotype.

We could prove in our in vitro studies that Bi-mAb-activated lymphocytes secreted enhanced levels of cytokines but that the tumor eradication was dependent mainly on the release of perforin and granzymes. This granule-exocytosis pathway seems to play a critical role in the present in vivo SCID mouse model, too. Only SCID mice treated with a combination of both Bi-mAbs had up-regulated mRNA expression of perforin and granzymes in the tumor tissue. The expression pattern was strictly CD30-antigen dependent. These data support former results of several studies investigating tumor cell lysis by effector cells. Mice lacking the perforin gene provided clear evidence that pore formation by perforin is the main mechanism of cytotoxicity mediated by CTLs and natural killer cells and is crucial for tumor destruction in vivo (33 , 34) . An approach similar to the one used by our group showed that TILs activated ex vivo by an anti-CD3-moAb and IL-2 expressed high levels of perforin, granzyme, and cytokine message. Moreover, up-regulation of these molecules in TILs correlated directly with the survival rate of tumor-bearing animals (27) . Recent trials (35) confirmed the correlation between up-regulation of perforin/granzymes in TILs and clinical response. In patients suffering from follicular lymphoma, TILs displayed high levels of perforin, which was interpreted as a marker of cytotoxic activity and was speculated to be caused by the endogenous local secretion of IL-2 by the tumor cells (35) . A similar result was published for patients with metastatic melanoma. In these patients, s.c. administration of IL-2 enhanced the pool of circulating perforin- or granzyme-positive lymphocytes in a dose-dependent fashion and modulated tumor-specific cytotoxicity (36) .

Our results underline the major advantage of the combined anti-CD3/anti-CD28 Bi-mAb approach by inducing a tumor site-specific activation of the T-cell cytolytic molecular program in vivo. The granule exocytosis model does not depend on receptor-ligand interactions and, in contrast to the FAS/APO-1 system, is not limited to FAS/APO-1-positive cells. Therefore, the described in vivo findings of Bi-mAb-mediated cytotoxicity should be transferable to a wide range of tumors irrespective of their FAS/APO-1 antigen status. As Bi-mAb-targeted and -activated lymphocytes did not home to TAA-negative tissues, the extent of side effects (caused e.g., by damage of normal tissue by activated T cells) should be low.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant PF 135/3-2 from the Deutsche Forschungsgemeinschaft (to M. P.). Back

2 To whom requests for reprints should be addressed, at Universität des Saarlandes, Kirrbergstrasse, D-66421 Homburg, Germany. Phone: 0049-6841/163084; Fax: 0049-6841/163004. Back

3 The abbreviations used are: Bi-mAb, bispecific monoclonal antibody; TAA, tumor-associated antigen; SCID, severe combined immunodeficient/immunodeficiency; moAb, monoclonal antibody; TNF, tumor necrosis factor; IL, interleukin; FACS, fluorescence-activated cell sorting; RT, reverse transcription; TIL, tumor-infiltrating lymphocyte. Back

Received 9/21/98. Accepted 2/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pardoll D. M. Cancer vaccines. Immunol. Today, 14: 310-316, 1993.[Medline]
  2. Greenberg P. D. Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells. Adv. Immunol., 49: 281-355, 1990.
  3. Renner C., Pfreundschuh M. Tumor therapy by immune recruitment with bispecific antibodies. Immunol. Rev., 145: 179-209, 1995.[Medline]
  4. Demanet C., Brissinck J., de Jonge J., Thielemans K. Bispecific antibody mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28 induced costimulation. Blood, 87: 4390-4398, 1996.[Abstract/Free Full Text]
  5. Milstein C., Cuello A. C. Hybrid hybridomas and their use in immunohistochemistry. Nature (Lond.), 305: 537-540, 1983.[Medline]
  6. June C. H., Ledbetter J. A., Linsley P. S., Thompson C. B. Role of the CD28 receptor in T-cell activation. Immunol. Today, 11: 211-216, 1990.[Medline]
  7. Jung G., Ledbetter J. A., Müller-Eberhard H. J. Induction of cytotoxicity in resting human T-lymphocytes bound to tumor cells by antibody heteroconjugates. Proc. Natl. Acad. Sci. USA, 84: 4611-4615, 1987.[Abstract/Free Full Text]
  8. Möller S. A., Reisfeld R. A. Bispecific monoclonal antibody directed lysis of ovarian carcinoma cells by activated human T-lymphocytes. Cancer Immunol. Immunother., 33: 210-216, 1991.
  9. Renner C., Jung W., Sahin U., Denfeld R., Pohl C., 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 (Washington DC), 264: 833-835, 1994.[Abstract/Free Full Text]
  10. Renner C., Bauer S., Sahin U., Jung W., van Lier R., Jacobs G., Held G., Pfreundschuh M. Cure of disseminated xenografted human Hodgkin’s tumors by bispecific monoclonal antibodies and human T-cells: the role of lymphocyte subsets in a preclinical model. Blood, 87: 2930-2937, 1996.[Abstract/Free Full Text]
  11. Qin Z., Richter G., Schuler T., Ibe S., Cao X., Blankenstein T. B cells inhibit induction of T cell-dependent tumor immunity. Nat. Med., 5: 627-630, 1998.
  12. Quian J-H., Titus J. A., Andew S. M., Mezzanzanica D., Garrido M. A., Wunderlich J. R., Segal D. M. Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth. J. Immunol., 146: 3250-3256, 1991.[Abstract]
  13. Beun G. D. M., van de Velde J. H., Fleuren G. J. T-cell based cancer immunotherapy: direct or redirected tumor-cell recognition?. Immunol. Today, 15: 11-14, 1994.[Medline]
  14. Nakamura Y., Wakimoto H., Abe J., Kanegae Y., Saito I., Aoyagie M., Kikiyoshi H., Hamada H. Adoptive immunotherapy with murine tumor-specific T lymphocytes engineered to secrete interleukin 2. Cancer Res., 54: 5757-5760, 1994.[Abstract/Free Full Text]
  15. Young J. D. E. Killing of target cells by lymphocytes: a mechanistic view. Physiol. Rev., 69: 250-314, 1989.[Free Full Text]
  16. Griffiths G. M. The cell biology of CTL killing. Curr. Opin. Immunol., 21: 687-692, 1995.
  17. Nakajima H., Henkart P. A. Cytotoxic lymphocyte granzymes trigger a target cell internal disintegration pathway leading to cytolysis and DNA breakdown. J. Immunol., 152: 1057-1063, 1994.[Abstract]
  18. Renner C., Held G., Ohnesorge S., Bauer S., Gerlach K., Pfitzenmeier J.-P., Pfreundschuh M. Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T-cells. Cancer Immunol. Immunother, 44: 70-76, 1997.[Medline]
  19. Renner C., Held G., Ohnesorge S., Bauer S., Gerlach K., Pfitzenmeier J-P., Pfreundschuh M. Role of naive and memory T cells in tumor cell lysis mediated by bispecific antibodies. Immunobiology, 197: 122-132, 1997.[Medline]
  20. Klein-Schneegans A. S., Gaveriaux C., Fonteneau P., Loor F. Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses. J. Immunol. Methods, 119: 117-125, 1989.[Medline]
  21. Habu S., Fukui H., Shimamura K., Nagai Y., Okumura K., Tamaoki N. In vivo effects of anti-asialo GM1. J. Immunol., 127: 34-38, 1981.[Abstract]
  22. Renner C., Jung W., van Lier R., Sahin U., Pfreundschuh M. The role of lymphocyte subsets and adhesion molecules in T-cell dependent cytotoxicity mediated by bispecific CD3 and CD28 bispecific monoclonal antibodies. Eur. J. Immunol., 25: 2027-2035, 1995.[Medline]
  23. Diehl V., von Kalle C., Fonatsch C., Tesch H., Jüecker M., Schaadt M. The cell of origin in Hodgkin’s disease. Semin. Oncol., 17: 660-672, 1990.[Medline]
  24. Schmitz J., Assenmacher M., Radbruch A. Regulation of T helper cell cytokine expression: functional dichotomy of antigen-presenting cells. Eur. J. Immunol., 23: 191-199, 1993.[Medline]
  25. Renner C., Ohnesorge S., Held G., Bauer S., Jung W., Pfitzenmeier J-P., Pfreundschuh M. T cells from patients with Hodgkin’s disease have a defective T-cell receptor {zeta} chain expression that is reversible by T-cell stimulation with CD3 and CD28. Blood, 88: 236-241, 1996.[Abstract/Free Full Text]
  26. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987.[Medline]
  27. Nakajima F., Khanna A., Xu G., Lagman M., Haschemeyer R., Mouradia J., Wang J. C., Stenzel K. H., Rubin A. L., Suthanthiran M. Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin-2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression. Proc. Natl. Acad. Sci. USA, 91: 7889-7893, 1994.[Abstract/Free Full Text]
  28. Thompson C. B., Lindstein T., Ledbetter J. A., Kunkel S. L., Young H. A., Emerson S. G., Leiden J. M., June C. H. CD28 activation pathway regulates the production of multiple T-cell derived lymphokines/cytokines. Proc. Natl. Acad. Sci. USA, 86: 1333-1337, 1989.[Abstract/Free Full Text]
  29. van Dijk J., Zegveld S. T., Fleuren G. J., Warnaar S. O. Localisation of monoclonal antibody G250 and bispecific monoclonal antibody CD3/G250 in human renal cell carcinoma xenografts: relative effects of size and affinity. Int. J. Cancer, 48: 738-743, 1991.[Medline]
  30. De Gast G. C., Van Houten A. A., Haagen I. A., Klein S., De Weger R. A., van Dijk A., Phillips J., Clark M., Bast B. J. Clinical experience with CD3 x CD19 bispecific antibodies in patients with B cell malignancies. J. Hematother., 5: 433-437, 1995.
  31. Klein S. C., Boer L. H., de Weger R. A., de Gast G. C., Bast E. J. Release of cytokines and soluble cell surface molecules by PBMC after activation with the bispecific antibody CD3 x CD19. Scand. J. Immunol., 46: 452-458, 1997.[Medline]
  32. Azuma M., Cayabyab M., Philips J. H., Lanier L. Requirement for CD28-T cell mediated cytotoxicity. J. Immunol., 150: 2091-2099, 1993.[Abstract]
  33. Kägi D., Ledermann B., Bürki K., Seiler P., Odermatt B., Olsen K. J., Podack E. R., Zinkernagel R. M., Hengartner H. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature (Lond.), 369: 31-32, 1994.[Medline]
  34. Walsh C. M., Maltoubian M., Liu C. C., Ueda R., Kurahara C. G., Christensen J. L., Huang M. T. F., Young J. D. E., Ahmed R., Clark W. R. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci USA, 91: 10854-10856, 1994.[Abstract/Free Full Text]
  35. Lèger-Ravet M. B., Devernge O., Peuchmaur M., Solal-Celigny P., Brousse N., Gaulard P., Galanaud P., Emilie D. In-situ detection of activated cytotoxic cells in follicular lymphomas. Am. J. Pathol., 144: 492-499, 1994.[Abstract]
  36. Lèger-Ravet M. B., Mathiot C., Portier A., Brandely M., Galanaud P., Fridman W. H., Emilie D. Increased expression of perforin and granzyme B genes in patients with metastatic melanoma treated with recombinant interleukin-2. Cancer Immunol. Immunother., 39: 53-58, 1994.[Medline]



This article has been cited by other articles:


Home page
 J BiochemHome page
X.-B. Wang, B.-F. Zhao, Q. Zhao, J.-H. Piao, J. Liu, Q. Lin, and H.-L. Huang
A New Recombinant Single Chain Trispecific Antibody Recruits T Lymphocytes to Kill CEA (Carcinoma Embryonic Antigen) Positive Tumor Cells In Vitro Efficiently
J. Biochem., April 1, 2004; 135(4): 555 - 565.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Bauer, N. Adrian, B. Williamson, C. Panousis, N. Fadle, J. Smerd, I. Fettah, A. M. Scott, M. Pfreundschuh, and C. Renner
Targeted Bioactivity of Membrane-Anchored TNF by an Antibody-Derived TNF Fusion Protein
J. Immunol., March 15, 2004; 172(6): 3930 - 3939.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. M. Kipriyanov, B. Cochlovius, H. J. Schafer, G. Moldenhauer, A. Bahre, F. Le Gall, S. Knackmuss, and M. Little
Synergistic Antitumor Effect of Bispecific CD19 x CD3 and CD19 x CD16 Diabodies in a Preclinical Model of Non-Hodgkin's Lymphoma
J. Immunol., July 1, 2002; 169(1): 137 - 144.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bauer, S.
Right arrow Articles by Pfreundschuh, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bauer, S.
Right arrow Articles by Pfreundschuh, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online