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The Journal of Immunology, 1998, 161: 1454-1461.
Copyright © 1998 by The American Association of Immunologists

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 Model1

Jan De Jonge*, Carlo Heirman*, Marijke de Veerman*, Sonja Van Meirvenne*, Muriel Moser{dagger}, Oberdan Leo{dagger} and Kris Thielemans2,*

* Vrije Universiteit Brussel, Medical School, Laboratory of Physiology-Immunology, Brussels, Belgium; and {dagger} Université Libre de Bruxelles, Laboratoire de Physiologie Animale, St-Genesius-Rode, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As demonstrated in several preclinical models, bispecific Abs are attractive immunotherapeutic agents for tumor treatment. We have previously reported that a bacterially produced anti-CD3 x antitumor bispecific single chain variable fragment of Ab fragment (BsscFv), which is capable of retargeting CTLs toward BCL1 tumor cells, exhibits antitumor activity in vitro. To further facilitate BsscFv production, the coding sequence was subcloned in a eukaryotic expression vector and introduced into Chinese hamster ovary cells for large-scale production. In this report, we have determined the serum stability and the clearance rate from the circulation of BsscFv. Most important, we prove here the therapeutic value of BsscFv in the treatment of BCL1 lymphoma, a murine model for human non-Hodgkin’s lymphoma. Tumor-bearing mice that were treated with rscFv in combination with staphylococcal enterotoxin B superantigen, human rIL-2, or murine rIL-12 showed long-term survival, whereas untreated mice all died. This is the first report of the successful in vivo use of BsscFv as an immunotherapeutic agent. Furthermore, long-term survival was the result of complete tumor removal and was not due to the induction of dormancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bifunctional Abs are being considered as potential therapeutic tools for the fight against neoplastic and virally infected cells. Such Abs are built up by two moieties. One part specifically recognizes an Ag on the target cells to be destroyed. The second part can be either a toxic compound (immunotoxin), a radioactive compound, or an Ab (fragment) recognizing an Ag or hapten (reviewed in Refs. 1-3). The latter types of bifunctional proteins are called bispecific Abs (BsAbs)3. If the second specificity is oriented against a marker that is present on effector cells of the immune system (i.e., CD16 on NK cells, CD3 on T cells), an immune effector cell retargeting toward the tumor cells can be achieved. The BsAb makes a bridge between the two cell types and triggers the immune cells to exert their effector function. The effectiveness of such an approach has been documented in vitro and in vivo in two animal models in our laboratory (4, 5). Several clinical phase I/II trials with BsAbs show little to moderate toxicity (6, 7). BsAbs can be produced in a number of ways. The first proteins were made by chemical cross-linking (8). In a second approach, BsAbs can be purified from the culture supernatant of hybrid-hybridomas, which are obtained by the fusion of two hybridoma cell lines that secrete Abs with the desired specificity (9). Since a complex mixture of heavy and light chain combinations is produced by such cell lines, a complex purification protocol is needed. Therefore, many investigators started to prepare BsAbs that are constructed by recombinant means, in which one recombinant protein with a dual specificity is produced. The development of smaller Ab fragments, such as bispecific single chain variable fragment of Ab fragments (BsscFvs) (10-15) and diabodies (16), which retain the parental specificity, offers several advantages over intact Ab molecules for therapeutic use. While BsscFv is built up by a single polypeptide chain, the diabody is formed upon the association of two polypeptides. The smaller proteins have a higher penetrating capacity into solid tissue (17), a lower toxicity/immunogenicity due to the lack of an Fc region (often evoking a human anti-mouse Ab response in human therapy, 7), and, probably, an absence of the immunosuppression observed with whole Abs (18). To decrease the human anti-mouse Ab response, efforts have been undertaken to construct chimeric human-mouse Abs (19-20). These smaller Ab fragments can be expressed in bacteria (10-14, 16), mammalian expression systems (i.e., Chinese hamster ovary (CHO) cells) (15), insect cells (21, 22), or yeast cells (23). In vitro data are available for the biologic activity of both diabodies and BsscFvs, proving their usefulness in the retargeting of selected cell types (10-15, 24). Recently, a method has been described for the generation of a large number of BsAbs using phage display repertoires (25).

Previously, we have shown that B cell lymphoma BCL1-bearing mice could successfully be treated with hybrid-hybridoma-derived BsAbB1x7D6 (4). The Ab can simultaneously bind to the CD3 marker on T cells (via the 7D6 portion) and to the Id of the surface-expressed Ig of BCL1 cells (via the B1 portion). Because this Id is a tumor-specific Ag, only these tumor cells will be recognized by the BsAb.

We have recently reported the construction of a BsscFv (BsscFvB1-2C11) with the same dual specificity (i.e., anti-Id and anti-CD3) (13). The recombinant protein was expressed in Escherichia coli in an insoluble form and refolded and reoxidized in vitro. The protein scored well in an in vitro CTL-retargeting assay, which subsequently allowed us to test its therapeutic potential in vivo in BCL1 tumor-bearing BALB/c mice.

To facilitate the production of the BsscFvB1-2C11 Ab fusion protein, we have now cloned the protein-coding gene in a eukaryotic expression vector for secretion by CHO cells. We report that the BsscFvB1-2C11, which is produced in bacteria as well as in CHO cells, can be successfully applied in vivo for the therapy of murine BCL1 lymphoma. Mice treated with BsscFvB1-2C11 (anti-Id x anti-CD3) show extended survival compared with control animals. When staphylococcal enterotoxin B (SEB), human rIL (rhIL)-2, or murine rIL (rmIL)-12 were included in the treatment, a higher survival rate was observed.

The beneficial effect of the SEB superantigen and IL-2 on the therapeutic outcome of BsAb-mediated therapy has been demonstrated before by other groups (26-27). These immunomodulatory molecules induce systemic T cell activation. IL-12 is another candidate molecule for increasing the number of activated T cells that can be recruited in the treatment. IL-12 is a heterodimeric cytokine with multiple effects on the immune system (reviewed in 28 . One of these effects is the enhancement of the proliferation of activated T cells (29) and of the cytotoxic activity of T lymphocytes (30). Since the antitumor effect of anti-CD3 x anti-tumor BsAbs is mainly the result of CTL retargeting in an MHC-unrestricted way, we found it worthwhile to test whether rmIL-12 is able to increase the survival of BCL1-bearing mice that had been treated with BsscFvB1-2C11 (31). In the case of NK cell-mediated therapy, the coadministration of IL-12 and anti-CD16 x antitumor improved the therapeutic outcome (32). In this report, we show that rmIL-12 does indeed augment the curative effect of the cytotoxic T cell-retargeting approach. In addition, we show that the induction of dormancy, which is a well-characterized phenomenon in the BCL1 model system (33), is not involved in long-term survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice strains, cell lines, and Abs

BALB/c mice were purchased from Charles River Wiga (Sulzfeld, Germany). Female mice were 8 to 12 wk old when used in therapy experiments.

BCL1 is a BALB/c-derived lymphoma cell line (34). An in vitro-adapted BCL1 cell line (BCL1vitro) was obtained from Dr. J.R. Parnes (Stanford University, Palo Alto, CA). This cell line stains positively with CTLA-4 Ig. Both cell lines express surface IgM{lambda}, which reacts with the anti-Id mAb B1 (IgG1K).

A second B cell lymphoma line of BALB/c origin, A20 (35), was used as a control cell line in the in vitro T cell proliferation and cytotoxicity assays.

Hybrid-hybridoma-derived BsAbB1x7D6 was constructed and purified as described previously (4).

Construction of eukaryotic expression plasmid pEE14-BsscFvB1-2C11

The construction and expression of BsscFvB1-2C11 in E. coli, as well as its purification and refolding, were as described previously (13). In short, the BsscFvB1-2C11 recombinant protein consists of VHB1, VLB1, VH2C11, and VL2C11, from the N terminus to the C terminus. All neighboring protein domains are connected by the (Gly4-Ser)3 peptide linker, resulting in a single polypeptide chain.

For eukaryotic expression, the BsscFvB1-2C11 gene was cloned into the pEE14 vector (36). The insert from the bacterial expression vector pQE-BsscFvB1-2C11 (13) was used to make the eukaryotic expression plasmid pEE14-BsscFvB1-2C11. The pelB signal sequence (37) upstream of the BsscFvB1-2C11 gene in the bacterial expression construct was replaced by the autologous leader sequence of the VH of the B1 Ab (anti-Id). The EcoRI/BspEI fragment containing the leader and part of the VHB1 cDNA was ligated together with the BspEI/HinDIII-restricted BsscFvB1-2C11 fragment from pQE-BsscFvB1-2C11. The EcoRI/HinDIII DNA fragment was ligated in the pEE14 vector that had been digested with EcoRI/HinDIII. Correctly assembled plasmids were identified by restriction enzyme digestion. pEE14 can be transiently expressed in COS-7 cells and is stable in CHO cells. The secreted BsscFvB1-2C11 protein was purified on a BCL1-Sepharose column and analyzed by SDS-PAGE.

In vitro serum stability and in vivo clearance of BsscFvB1-2C11

To determine the stability of hybrid-hybridoma-derived BsAbB1x7D6 and rBsscFvB1-2C11, 10 µg of the protein was incubated in 100% filter-sterilized normal mouse serum at 37°C. At several time intervals, an aliquot was removed and frozen at -20°C until tested. The remaining biologically active BsAb was measured by a standard T cell proliferation assay as described below.

To investigate the in vivo clearance from the serum, naive BALB/c mice were i.v. injected with 40 µg of BsscFvB1-2C11 or BsAbB1x7D6 into the tail vein. The mice were bled at various time points, and the presence of BsAb in their serum was analyzed using a T cell proliferation assay. The activity present at 2 min postinjection was used as timepoint 0 activity.

Standard molecular and cellular techniques

Restriction digestion, DNA purification, ligation, agarose electrophoresis, and SDS-PAGE were performed according to standard procedures (38). rbsscFvB1-2C11 protein was transiently expressed in COS-7 cells using the DEAE-dextran transfection method. The stable transfection of CHO cells was performed by lipofection as described previously (38).

For the T cell proliferation assay, BCL1vitro or A20 cells were incubated with mitomycin C (50 µg/ml) for 90 min or 6 h, respectively, at 37°C in the dark. After extensive washing to remove excess mitomycin C, the tumor cells were plated at 5 x 104 cells/well and cocultured in standard medium with 1 x 105 syngeneic BALB/c spleen cells from naive mice in the presence or absence of the indicated concentration of bispecific protein. After 2 days, the cellular proliferation was measured by pulsing with 0.5 µCi of [3H]TdR (1 mCi/ml, Amersham, Little Chalfont, U.K.) for 18 h before the cells were harvested with an automated cell harvester. The incorporated radioactivity was measured by scintillation counting (Microbeta, Wallac, Berthold, Vilvoorde, Belgium).

For the redirected cytotoxicity assay, primary, alloreactive CTLs were generated as described previously (4). Briefly, 4 x 106 splenic responder cells (BALB/c) were cultured for 5 days with 2 to 4 x 106 mitomycin C-treated (50 µg/ml for 1 h at 37°C) stimulator cells (C57BL/6) in 2 ml of complete medium supplemented with 10 U/ml rhIL-2. The BCL1vitro or A20 cells were 51Cr-labeled by incubating 2 x 106 cells with 150 µCi of Na251CrO4 (Amersham) in 500 µl of RPMI 1640 supplemented with 5% FCS for 1 h at 37°C. The labeled target cells were washed three times before use. Effector cells and labeled target cells were added to V-bottom wells in triplicate at an E:T ratio of 50:1 and a final volume of 200 µl. The 51Cr released from the target cells was measured in a standard 4-h assay in the presence of BsscFvB1-2C11 fusion protein or hybrid-hybridoma-produced BsAbB1x7D6. Cell lysis was assessed in a gamma counter (GAMMAmatic I, Kontron Analytical, van Hopplynus, Brussels, Belgium). The percentage of specific lysis was calculated as follows: ([Ecpm - Scpm]/[Mcpm - Scpm]) x 100; where Ecpm = 51Cr released from the target cells plus effector cells in the presence of Abs, Mcpm = release from target cells in 0.05 M hydrochloric acid, and Scpm = spontaneous release of 51Cr from the target cells in the medium in the absence of effector cells. The Scpm was never >20% of the maximum release.

Therapy experiments

For therapy experiments, mice were injected i.p. with the indicated number of BCL1 lymphoma cells in PBS. During the treatment, BsAb was injected i.v. into the tail vein; SEB, rhIL-2, or rmIL-12 was injected i.p. at the indicated dose. SEB and rhIL-2 were from Sigma (St. Louis, MO) and Cetus (Emeryville, CA), respectively. rmIL-12 was purified by immobilized metal chelate affinity chromatography as described in Reference 39.

Dormancy experiments

After BsAb treatment, we attempted to identify circulating BCL1 IgM in the serum of surviving mice to test for the presence of tumor cells. Therefore, mouse serum was incubated on B1-coated 96-well ELISA plates; bound BCL1 was then detected with peroxidase-labeled goat-anti-mouse IgM according to standard procedures (4). Several mice that were tumor free at 120 days after BsAb therapy (as assessed by the absence of circulating BCL1 IgM in their serum), were sacrificed, and spleen cells were isolated. Macrophages and fibroblasts were removed by plastic adherence; T cells were removed by panning on anti-CD3 (145-2C11)-coated petri dishes. Enriched B cells (107) were injected i.p. into naive BALB/c mice that had been irradiated with 450 rad 2 days earlier. Cell depletion was performed to prevent an eventual cotransfer of dormancy-sustaining cellular immunity (see Ref. 33 for review on dormancy).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of BsscFvB1-2C11

Initially, BsscFvB1-2C11 was expressed in bacterial cells (13) in an insoluble form and was refolded and reoxidized in vitro. When the biologic activity of purified BsscFvB1-2C11 was assayed in a 51Cr release CTL test, batch-to-batch differences were observed (data not shown). This was most likely due to improper folding or disulfide bond formation in the anti-CD3 scFv moiety of the bispecific product. The SDS-PAGE analysis shown in Figure 1GoA demonstrates that, although only one band appeared when BsscFvB1-2C11 was electrophoresed under reducing conditions, BsscFvB1-2C11 on a nonreducing SDS-PAGE showed a second band with slightly different gel mobility. This observation indicates that incorrect S-S bridges were formed in addition to the correct disulfide bonds. To avoid the unpredictable and inefficient step of refolding of the bacterially produced BsscFvB1-2C11, we subcloned the BsscFvB1-2C11 gene in the pEE14 vector for the expression in CHO cells (see Materials and Methods). The functionality of the resulting pEE14-BsscFvB1-2C11 plasmid was confirmed in a transient COS-7 expression system followed by a T cell proliferation assay. Figure 2Go illustrates that spleen T cells from naive animals are activated only when the BsscFv was present in combination with BCL1 cells but not when A20 cells (BCL1-Id-negative) are present. Thus, the BsscFvB1-2C11 protein expressed from the pEE14-BsscFvB1-2C11 plasmid is functionally active and specific for BCL1 tumor cells. The pEE14-BsscFvB1-2C11 vector was then introduced by transfection into CHO cells, which are used for the large-scale production of the recombinant protein. The BsscFvB1-2C11 fusion protein was secreted in the culture supernatant, purified by affinity chromatography, and analyzed by SDS-PAGE (Fig. 1GoB). Since the fusion protein migrated as a single band under reducing as well as nonreducing conditions, we can assume that only correct S-S bridges were formed. The yield of purified BsscFvB1-2C11 was ~5 mg per wk per liter of culture supernatant. A total of 1 L of supernatant corresponds with 5250 cm2 of a confluent monolayer of CHO cells. Thus, ~1 µg of BsscFvB1-2C11 is produced by 1 cm2 of cells in 1 wk.



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  		FIGURE 1. SDS-PAGE of BsscFvB1-2C11. Bacterially produced and in vitro-refolded (A) or CHO-produced (B) BsscFvB1-2C11 was affinity purified and analyzed on a nonreducing (NR) and a reducing (R) gel. M is the m.w. standard.

 


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  		FIGURE 2. T cell proliferative activity of rBsscFvB1-2C11. COS-7 cells were transfected with pEE14-BsscFvB1-2C11. After 8 days, the supernatant was tested for the presence of BsscFvB1-2C11 by a T cell proliferation assay in the presence of BCL1 or A20 cells. BsscFvB1-2C11, which had been purified from CHO cells, was at a concentration of 1 µg/ml at a 1/1 dilution.

 
Before in vivo experiments were initiated, the biologic activity of the BsscFvB1-2C11 was tested in a T cell proliferation assay (Fig. 2Go) and a CTL-retargeting assay (Fig. 3Go). Both tests indicate that the dual specificity and the biologic activity of the recombinant fusion protein are preserved.



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  		FIGURE 3. Redirected tumor cell lysis by CHO-produced BsscFvB1-2C11. Labeled target cells (BCL1 or A20) were lysed by allogeneic CTLs in the presence of the indicated concentration of BsAbB1x7D6 (hybrid-hybridoma) or BsscFvB1-2C11 (CHO) in a 4-h standard cytotoxicity assay. The E:T cell ratio was 50:1.

 
In vitro serum stability and in vivo clearance of BsscFvB1-2C11

The stability of BsscFvB1-2C11 fusion protein in normal mouse serum was compared with that of hybridoma-derived BsAbB1x7D6. A 4-h incubation resulted in 0% (BsAbB1x7D6) and 10% (BsscFvB1-2C11) activity loss. After a 24-h incubation, the BsAbB1x7D6 retained 90% of its starting activity. In contrast, the BsscFvB1-2C11 kept only 25% of its original biologic activity, as determined in a T cell proliferation assay (Fig. 4GoA).



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  		FIGURE 4. Stability of BsscFvB1-2C11. The stability of BsAbB1x7D6 and BsscFvB1-2C11 in mouse serum (A) and the serum clearance (B) was compared. A, BsAbs were incubated in sterile normal mouse serum at 37°C at a concentration of 10 µg/ml, and aliquots were removed at different time points. B, A total of 40 µg of BsAb was i.v. injected into naive mice. Serum from the animals was prepared at different time points. The biologic activity in the serum samples was assessed by T cell proliferation. The activity of the samples at timepoint 0 (0 min for A; 2 min for B) was taken as 100% activity.

 
Next, we tested the clearance of both proteins from the circulation after i.v. injection in mice (Fig. 4GoB). The t1/2 of BsscFvB1-2C11 was 30 min; after 4 h, its activity was below the detection limit of the assay. In the case of BsAbB1x7D6 protein, 80% of the protein was still present in the circulation at 4 h postinjection.

Treatment of BCL1-bearing mice with BsscFvB1-2C11

In vivo experiments were performed using both bacterially and CHO-produced BsscFvB1-2C11 fusion protein. In Figure 5Go, the therapeutic potential of BsAbB1x7D6 and BsscFvB1-2C11 are compared. Mice received 5000 BCL1 cells (i.p.) on day 0 and were treated 9 days later with an i.v. injection of either 5 µg of BsAbB1x7D6 (n = 8) or BsscFvB1-2C11 (n = 8). Only BsAbB1x7D6-treated animals (62%) survived to 100 days posttreatment. All untreated and BsscFvB1-2C11-treated mice died. Repeating the BsscFvB1-2C11 administration four times with the 5-µg dose did not lead to long-term survival. Only a small shift in the survival rate could be observed. Increasing the amount of BsscFvB1-2C11 per injection led to the conclusion that four injections of 20 µg was optimal; higher doses did not result in a substantial increase in the survival percentage (data not shown). Therefore, 20 µg of BsscFvB1-2C11 per injection was used in the experiments described below. The accumulated data from several repeat experiments are presented. A total of 5000 BCL1 tumor cells were inoculated i.p. on day 0. The animals were given a single 5-µg dose of hybrid-hybridoma-produced BsAbB1x7D6 (20 mice) on day 9 or 20 µg of BsscFvB1-2C11 (20 mice, once a day from days 9-12) by i.v. injection in the tail vein. All mice that were treated with a mixture of monospecific scFvB1 and scFv2C11 died. A total of 50% of the animals treated with BsAbB1x7D6 and 25% of the BsscFvB1-2C11-treated group survived to 200 days after tumor inoculation (Fig. 6Go).



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  		FIGURE 5. Therapy of BCL1-bearing mice with BsAbs. BALB/c mice received 5000 BCL1 cells at day 0 and were treated with a single dose of 5 µg of BsAbB1x7D6 or BsscFvB1-2C11 (n = 8; injected i.v. at day 9), four doses of 5 µg of BsscFvB1-2C11 (n = 8; injected i.v. from days 9-12), or left untreated (n = 8).

 


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  		FIGURE 6. rBsscFvB1-2C11 induces the long-term survival of BCL1 tumor-bearing mice. BALB/c mice were injected i.p. with 5000 BCL1 cells at day 0. The mice were treated with 5 µg of hybrid-hybridoma-derived BsAbB1x7D6 (n = 20; injected i.v. at day 9), four doses of 20 µg of BsscFvB1-2C11 (n = 20; injected i.v. from days 9-12), or four doses of 10 µg of scFvB1 and scFv2C11 each (n = 5; injected i.v. from days 9–12).

 
Influence of T cell activators on BsscFvB1-2C11 therapy

Several studies have indicated the importance of T cell stimulation in the treatment of tumors using BsAbs. It has been shown that the effect of anti-CD3 x anti-Id-based therapy could be drastically enhanced if it is combined with concomitant T cell stimulation (27). This stimulation could be achieved by either IL-2 or SEB superantigen treatment. To investigate the importance of activation by IL-2 or SEB in the case of BsscFvB1-2C11 therapy, we designed a treatment schedule as follows: BALB/c mice (n = 20) that had been inoculated with 5.103 BCL1 cells were injected on day 8 with 50 µg of SEB, followed by four daily i.v. injections of 20 µg of BsscFvB1-2C11. As illustrated in Figure 7Go, the combined treatment resulted in an increased survival rate (55% in comparison with 25% in the case of BsscFvB1-2C11 therapy alone) at day 200 after tumor injection. None of the mice treated with SEB alone (n = 14) survived.



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  		FIGURE 7. SEB increases the therapeutic effect of BsscFvB1-2C11. BALB/c mice were injected i.p. with 5000 BCL1 cells at day 0. The mice were treated with four doses of 20 µg of BsscFvB1-2C11 (n = 20; injected i.v. from days 9-12), with 50 µg of SEB (n = 14; injected i.p. at day 8), or with BsscFvB1-2C11 and SEB combined (n = 20).

 
To test the effect of T cell activation by IL-2, 20 mice were treated with 10,000 international units of rhIL-2 (twice a day for 4 days) and 20 µg of BsscFvB1-2C11 (four daily doses). While treating BCL1-bearing mice (n = 19) with rhIL-2 induced 15% survival as such, the combined therapy (rhIL-2 plus BsscFvB1-2C11) of 20 mice resulted in 80% survival at day 200 (Fig. 8Go). Thus, at least at the concentrations used in these experiments, rhIL-2 seems to be a more potent, nonspecific activator of T cells than SEB.



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  		FIGURE 8. IL-2 increases the therapeutic effect of BsscFvB1-2C11. BALB/c mice were injected i.p. with 5000 BCL1 cells at day 0. The mice were treated with four doses of 20 µg of BsscFvB1-2C11 (n = 20; injected i.v. from days 9–12), with 104 international units of rhIL-2 (n = 19; injected i.p. from days 7-10, twice a day), or with BsscFvB1-2C11 and rhIL-2 combined (n = 20).

 
Since IL-12 also leads to an activation of T cells, we wanted to test this cytokine with regard to its ability to augment the outcome of BsscFvB1-2C11 treatment in the BCL1 lymphoma model. Under the conditions used here, neither 10 ng nor 250 ng of rmIL-12 per injection induced any long-term survival of the tumor-bearing mice. However, when rmIL-12 (10 or 250 ng) was combined with BsscFvB1-2C11, 83% of mice survived compared with 50% when BsscFvB1-2C11 was used alone (Fig. 9Go).



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  		FIGURE 9. IL-12 increases the therapeutic effect of BsscFvB1-2C11. BALB/c mice were injected i.p. with 5000 BCL1 cells at day 0. The mice were treated with four doses of 20 µg of BsscFvB1-2C11 (n = 8; injected i.v. from days 9-12), with 10 ng or 250 ng of rmIL-12 (n = 6 for both quantities; injected i.p. from days 10-13), or with BsscFvB1-2C11 and rmIL-12 combined (n = 6 for both 10 ng and 250 ng of rmIL-12). Data from the treatment with 10 ng or 250 ng of rmIL-12 alone are presented together.

 
Mice treated with BsscFvB1-2C11 are free of dormant tumor cells

Finally, we wanted to investigate whether the surviving mice were free of dormant BCL1 cells. A total of 20 cured animals, as determined from the absence of circulating BCL1 IgM in the serum, were sacrificed. A total of 107 enriched splenic B cells from each of these 20 mice were transferred into two naive, irradiated BALB/c mice. None of these mice developed a tumor after 70 days. Instead, mice that had received B cells from tumor-bearing mice died within 60 days after cell transfer (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
On the condition that tumor-associated or tumor-specific Ags from a particular tumor have been identified, BsAbs can be used in the battle against the particular disease. Until recently, most BsAbs were created using hybrid-hybridomas. Nowadays, rBsAb fragments (such as Fabs, BsscFvs, and diabodies) that bridge target cells to effector cells can be expressed in E. coli. The biologic activity of such molecules in vitro is documented in several reports (10-15, 24). Theoretically, a scFv molecule can be selected for each Ag from a phage display library (40). scFvs and diabodies have an advantage over complete Abs in that they are smaller and, consequently, have better tissue penetration. Additionally, they lack the Fc portion, which can provoke an immune response if it is of xenogenic origin (7). Furthermore, we have previously determined that hybrid-hybridoma-derived BsAb caused a dose-related immunosuppression that could be mediated by Fc (18). This phenomenon limits the amount and frequency of the administration of BsAbs. Therefore, BsscFvs lacking the Fc portion will not possess this suppressive effect and should be superior immunotherapeutic molecules.

In this report of in vivo studies, we show that bacterially and CHO-produced rBsscFvB1-2C11 can both prolong the survival of and cure tumor-bearing mice. Although it is clear from our observations that BsscFvB1-2C11 has therapeutic effect in BCL1-bearing mice, this effect is less significant than that displayed by hybrid-hybridoma-produced BsAbB1x7D6. One possible explanation is that the anti-CD3 binding subunit of the two bispecific proteins originates from a different parental Ab. However, no major difference in the in vitro biologic activity of these two proteins was seen (13). Alternatively, the difference in the therapeutic outcome between BsAbB1x7D6 and BsscFvB1-2C11 might be explained by the much faster clearance of scFv Ab fragments from the circulation, as was previously shown by others (21) and illustrated by our own data (Fig. 4Go).

Another explanation for the difference in the therapeutic potential of both bispecific proteins could be the killing of the BCL1 cells by effector T cells plus BsAbB1x7D6-induced Ab-dependent cell-mediated cytotoxicity (ADCC). Indeed, it was previously demonstrated that ADCC, which are induced by the biisotypic BsAb (IgG1/G2a), resulted in some therapeutic effect when a low tumor load (500 BCL1 cells) was administered to the animals (4). However, when 5000 BCL1 cells were injected (as in the in vivo experiments described here), the effect of ADCC was completely abrogated.

The antitumor effect of BsAbs is mainly the result of CTL retargeting in an MHC-unrestricted way. The role of perforin and granzymes in CTL retargeting has been demonstrated previously (41). Compounds that increase the pool of cytotoxic effector cells are interesting to evaluate in this kind of immunotherapeutic approach.

It has been shown by several groups that the activation of T cells by bacterial superantigens (26), cytokines (IL-2 (26), IL-12 (31)), or costimulatory molecules (18, 27, 42, 43) can result in an increased therapeutic effect of BsAb therapy. Here, we show that the same result is seen in the BCL1 lymphoma model when BsscFvB1-2C11 is used in combination with SEB, rhIL-2, and rmIL-12.

Bacterial superantigens generate IL-2-responsive T cells (44). However, these cells undergo apoptosis in the absence of IL-2. In an attempt to avoid SEB-induced T cell apoptosis, we combined SEB and rhIL-2 with BsscFvB1-2C11. However, the therapeutic outcome did not improve as compared with the combination of SEB and BsscFvB1-2C11 (data not shown).

IL-12, which is also known as T cell-stimulating factor, is a promising molecule for antitumor therapy. Since it promotes a Th1 immune response, it plays a pivotal role in controlling cell-mediated immunity (28). Predominantly produced by monocytes/macrophages and dendritic cells, IL-12 exerts its effects by binding on the IL-12R that is present on activated T and NK cells, resulting in their proliferation. IL-12 can increase the cytotoxic activity of CTLs by augmenting the expression of lytic components in the effector cells (31). In a number of transplantable murine tumor models, the administration of IL-12 resulted in a tumor-free state. A rechallenge with tumor cells showed that a tumor-specific immunity had developed (45). However, rmIL-12 was not able to cure BCL1-bearing mice in the treatment schedule used in the experiments described in this report. An effect of rmIL-12 was only observed when rmIL-12 and BsscFvB1-2C11 were used in combination. Thus, in this model system, no tumor-specific immune response seemed to be induced by rmIL-12. Similar to our experiments, BsAb-mediated NK retargeting and in vitro tumor cell lysis can be increased when IL-12 is added to the assay (32).

The faster clearance of rBsAb compared with hybrid-hybridoma-derived BsAb cannot solely be explained by its faster activity loss in mouse serum. Indeed, these processes show different kinetics. Because of its smaller size, BsscFv can more easily leave the bloodstream and, consequently, can be distributed among the different tissues. We further investigated whether a fraction of BsscFvB1-2C11 was bound to the T cells in the spleen after injection. Indeed, when spleen cells from mice that had been injected with BsscFvB1-2C11 were coincubated with BCL1 cells, T cell proliferation was observed. Since no exogenous BsscFvB1-2C11 had been added to this assay, we concluded that part of the injected BsscFv was bound to the splenic T cells (data not shown).

Instead of a complete removal of tumor cells by BsAb therapy, the induction of dormancy might also lead to long-term survival (33). In the BCL1 lymphoma model, dormancy has been correlated with the presence of circulating anti-Id Abs, which presumably result in ADCC and cross-linking of the B cell receptor complex on the tumor cells and consequently lead to growth-inhibiting signaling. Although BsscFvs are unable to cross-link surface IgMs (each contains only one anti-Id arm), we still wanted to rule out this possibility. This state of dormancy can be tested by PCR (46) or by the transfer of "cured" spleen cells into naive animals (47). Since we frequently observed false positives with the PCR approach, we decided to address the dormancy question using cell transfer. Vitetta et al. (47) showed that, on average, 106 BCL1 cells (or slightly <1% of the total spleen cell number) remain present in the spleens of all animals with a dormant tumor during the time interval analyzed (2-7 mo). Since we used 107 spleen cells in our transfer experiments, the number of dormant BCL1 cells eventually transferred would be in the range of 105. This amount of tumor load would certainly have evolved into detectable BCL1 IgM circulation after 70 days. Since we did not observe this circulation, we conclude that the BsscFvB1-2C11-treated mice were cured, with no dormant BCL1 lymphoma cells left.

Diabodies, which were first described by Holliger (16), are an alternative to BsscFvs. The bispecific molecules are formed by two polypeptide chains which, upon association, form the two Ag-binding sites. A diabody possessing the same specificity as BsscFvB1-2C11 has been constructed and functionally characterized in vitro (24). Tumor lysis by retargeted CTLs demonstrated that, on a w/v basis, the diabody was 10 times more active than hybrid-hybridoma-derived BsAbB1x7D6. BsscFvB1-2C11 is about three times more active than BsAbB1x7D6. Expressed on a molar basis, BsscFvB1-2C11 and BsAbB1x7D6 show comparable activity; the diabody would then be three times stronger. Whether this is also the case in vivo remains to be shown.

Although BsAb can trigger the killing of the tumor cells it recognizes, Id variants are not eliminated and will probably lead to the outgrowth of a variant tumor. Heterogeneity in the lymphoma cell population has been described in patients and in mouse lymphoma models (48, 49). This finding implies that if the treatment of BsAbs aims at killing all tumor cells, a combination of BsscFvs recognizing different epitopes of the Id rather than a single BsAb will be necessary to minimize treatment escape by variant lymphoma cells. With the current technology of phage Ab display, Abs against different epitopes of an Ag should be easy to obtain and could be used for the construction of several BsscFvs. Another approach is the use of BsAbs which bind to a pan-B cell marker (CD19, CD22) instead of to a tumor-specific Ag (50). We will investigate whether the accompanying loss of specificity will be reflected in a lower therapeutic benefit. To that purpose, we have constructed an anti-CD19 x anti-CD3 BsscFv protein and compared its therapeutic effect in vivo with that of BsscFvB1-2C11.

Although high expression levels can be obtained in bacterial expression systems, the fact that recombinant proteins often accumulate in an insoluble form in inclusion bodies is a major drawback. This necessitates the in vitro refolding of the protein, which is a rather inefficient process that leads to a low protein recovery (~1% in the case of BsscFvB1-2C11) (13). Furthermore, the S-S bridge formation in our bispecific fusion protein was not entirely correct. Consequently, a fraction of the refolded protein has not adopted the right three-dimensional shape and will be nonfunctional. Although superior refolding procedures with an improved recovery of functional protein have been described previously (51), the application of such procedures did not result in a higher yield of properly folded BsscFvB1-2C11 in our study (our unpublished observations). To circumvent these problems, we have started the production of BsscFvB1-2C11 in eukaryotic cells. Using the CHO expression system, correctly folded and biologically functional BsscFvB1-2C11 can be purified from the spent culture medium. A total of 1 cm2 of confluently growing CHO cells yields ~1 µg of BsAb in 1 wk.

Considering the data presented here, we conclude that the use of BsscFv molecules can be considered for the removal of the residual tumor cells remaining after standard tumor therapy. To our knowledge, this is the first report on the successful use of BsscFvs in the treatment of tumors.


   Footnotes
 
1 This work was supported by a grant from the Fund for Scientific Research-Flanders (FWO), the Algemene Spaar-en Lijfrentekas, the Vereniging voor Kankerbestrijding, and the Ministry of Science. M.M. and O.L. are research associates at the National Fund for Scientific Research. Back

2 Address correspondence and reprint requests to Dr. K. Thielemans, Vrije Universiteit Brussel, Medical School, Laboratory of Physiology-Immunology, Laarbeeklaan 103/E, 1090 Brussels, Belgium. Back

3 Abbreviations used in this paper: BsAb, bispecific Ab; Fv, variable fragment of Ab; scFv, single chain variable fragment of Ab; BsscFv, bispecific single chain variable fragment of Ab; CHO, Chinese hamster ovary; hIL-2, human IL-2; mIL-12, murine IL-12; SEB, staphylococcal enterotoxin B; ADCC, Ab-dependent cell-mediated cytotoxicity. Back

Received for publication May 27, 1997. Accepted for publication April 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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