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The Journal of Immunology, 2003, 170: 2695-2701.
Copyright © 2003 by The American Association of Immunologists

Anti-idiotype x Anti-LFA-1 Bispecific Antibodies Inhibit Metastasis of B Cell Lymphoma1

Sharon Cohen, Joseph Haimovich and Nurit Hollander2

Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id x anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The process of metastasis requires detachment of neoplastic cells from the primary tumor, followed by migration of the cells into the vascular system and extravasation into target organs. These crucial steps of metastasis require efficient interactions of tumor cells with surrounding cells and with extracellular matrix, which are mediated by adhesion molecules. Lymphoid neoplasms display a wide spectrum of growth pattern, from limited spread to wide dissemination. Since normal lymphocytes circulate continuously and extravasate into lymphoid organs and inflamed tissues, it is conceivable that mechanisms of normal lymphocyte homing operate in dissemination of their malignant counterparts. Increasing evidence indicates that adhesion molecules, involved in normal lymphocyte trafficking, have an essential role in lymphoma dissemination (1, 2, 3). Moreover, adhesion molecules are involved in postextravasation events that control progression and growth after homing to the target organ (4). Based on these observations, it appears likely that Abs to adhesion receptors may block lymphoma dissemination. Indeed, it has been demonstrated that Abs directed against adhesion molecules can block lymphoma metastasis in experimental models (5, 6, 7, 8), emphasizing the clinical potential of such Abs. However, since normal cells express a variety of adhesion molecules as well, treatment with Abs against adhesion molecules may also block normal function (9, 10, 11).

To circumvent adverse effects of antiadhesion Abs on normal cells, we proposed the use of bispecific Abs that bind simultaneously to a particular adhesion molecule and to a tumor-specific Ag. Such bifunctional Abs are expected to bind more avidly to tumor cells that coexpress both target Ags than to normal cells that express only the adhesion receptor. The anticipated selectivity of the proposed bispecific Abs results from the preferential binding of bispecific Abs to cells that coexpress both Ags (12, 13). We have recently produced a bispecific Ab with specificity to the integrin LFA-1 (CD11a/CD18) and to the Id of the murine B cell lymphoma 38C-13 (14). The 38C-13 Id is a well-established, tumor-specific Ag that is commonly used by us and others as a target for specific immunotherapy (15, 16, 17, 18). Our in vitro studies demonstrated that the anti-Id x anti-LFA-1 bispecific Ab selectively blocks LFA-1-mediated adhesion of Id-expressing tumor cells, but has no effect on the adhesion of normal lymphocytes (14).

In the current study we investigated the in vivo effects of the bispecific Ab. We demonstrate that the Ab blocks lymph node and liver metastasis in mice carrying primary s.c. tumors. In contrast to anti-LFA-1 Abs, the bispecific Ab has no effect on in vivo immune responses. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave normal functions intact.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C3H/eB mice were obtained from the animal facility of Tel Aviv University and used at the age of 8–10 wk.

Cell lines and Abs

38C-13, a carcinogen-induced B cell lymphoma, was maintained as previously described (19). The hybridoma cell lines 2B6 (15) and D5D10 (unpublished) secrete rat mAb that are specific for an idiotypic determinant of the 38C-13 IgM. The 2B6 and D5D10 mAb were purified from ascites fluid by affinity chromatography as previously described (20). F(ab')2 of 2B6 were prepared by pepsin digestion. The purified mAb at a concentration of 1 mg/ml was digested at pH 4.4 with 4% pepsin (w/w) for 18 h at 37°C. The reaction was stopped by the addition of Tris base. The digestion mixture was analyzed by SDS-PAGE to verify complete digestion and purity of F(ab')2. The hybridoma cell line M17/4.4 (M17), secreting a rat mAb against the {alpha} subunit of murine LFA-1, was obtained from American Type Culture Collection (Manassas, VA). The M17 Ab was purified from ascites fluid by protein G-Sepharose fractionation. The anti-Id x anti-LFA-1 bispecific Ab was generated by somatic cell hybridization of the 2B6 and M17 cell lines (14). It was purified from ascites fluid using affinity chromatography and pH gradient elution as previously described (14).

Tumor inoculation and Ab treatment

Mice were inoculated s.c. with 1 x 105 38C-13 cells. At the indicated times, mice were injected i.p. with 0.2 mg of purified Abs.

Preparation of single-cell suspensions

Lymph nodes and spleens were dissociated by gentle mechanical homogenization through a nylon mesh. Livers were cut into small segments with scissors. The segments were then gently homogenized through a stainless still mesh, and the slurry was centrifuged once. The pellet was resuspended and incubated for 1 h at 37°C in digestion medium consisting of serum-free RPMI 1640, 0.1% (w/v) collagenase IV (Sigma-Aldrich, St. Louis, MO), and 0.002% (w/v) DNase I (Sigma-Aldrich). Malignant and normal lymphoid cells were then isolated from the suspension by density gradient centrifugation, using Lympholyte M (Cedarlane, Ontario, Canada) as the cell separation medium.

Determination of tumor metastasis

Tumor dissemination was determined by cell proliferation assays as previously described (21). Briefly, lymph node, spleen, and hepatic single-cell suspensions were incubated for 24 h at 1 x 105 cells/well in flat-bottom 96-well microplates. They were then pulsed for 20 h with 1 µCi/well of [3H]thymidine and harvested. Thymidine uptake was assessed by liquid scintillation counting. Under these conditions the presence of 38C-13 cells in lymph nodes and liver was manifested by extensive cell proliferation (high rates of thymidine uptake) compared with low proliferation rates in normal organs.

Dissemination of 38C-13 cells to different organs was also demonstrated by fluorescence staining of cell suspensions with the 2B6 anti-Id mAb, which is specific for the IgM of 38C-13. Flow cytometry was performed as previously described (21).

In addition, metastasis was determined by the weights of the different organs.

Determination of cell migration

In vivo migration of 38C-13 was determined by the radioactivity in different organs of mice injected i.v. with 51Cr-labeled cells. 38C-13 cells (2 x 107/ml) were incubated with 100 µCi of [51Cr]sodium chromate (PerkinElmer Life Sciences, Boston, MA) for 1 h at 37°C. Cells were then washed three times and injected i.v. into the tail vein (2 x 106 cells in 0.5 ml). For Ab inhibition determination, the cells were mixed with 0.2 mg of purified Abs. After 1 and 24 h, mice were sacrificed, and different organs were collected. The radioactivity of whole organs was counted in a gamma counter. The total radioactivity in blood was determined in 200-µl aliquots of blood and computed for a total volume of 2 ml of blood/mouse. Data are presented as the percentage of input counts in whole organs.

Sensitization and elicitation of delayed-type hypersensitivity (DTH)3

Twenty microliters of 1% 2,4-dinitro-1-fluorobenzene (DNFB; Sigma-Aldrich) in acetone/olive oil (4/1; Sigma-Aldrich) were painted on each footpad for 2 successive days. Five days later the mice were challenged by applying 2.5 µl of 1% DNFB in the same vehicle to each ear. Ear thickness was measured by caliper before challenge and 24 h postchallenge.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-Id x anti-LFA-1 bispecific Abs inhibit metastasis of 38C-13

When 38C-13 cells are inoculated s.c. or i.p. into mice, they grow at the site of inoculation and disseminate to different organs, including lymph nodes, spleen, lung, liver, and bone marrow (20, 21). Tumor dissemination is manifested by enlargement of the infiltrated organs, as determined by increases in organ weight and cell number. The presence of tumor cells in the different organs was determined by staining with tumor-specific anti-Id Abs. In moribund mice the majority of cells in affected organs are Id-positive tumor cells. In addition, tumor metastasis was determined by ex vivo assessment of cell proliferation. Cells derived from lymphoma-infiltrated tissues proliferate extensively ex vivo due to massive proliferation of the tumor cells, whereas cells of tumor-free tissues do not proliferate ex vivo.

To assess the effect of anti-Id x anti LFA-1 bispecific Abs on tumor metastasis, 38C-13-inoculated mice were treated with 0.2 mg of bispecific Abs 2 days after tumor inoculation and every 7 days thereafter. Mice were killed on day 21, when untreated mice became moribund, and the effect of Ab treatment was assessed by organ weight and thymidine uptake determination in cell cultures of several organs. The bispecific Ab treatment attenuated the growth of the s.c. primary tumors and reduced tumor load in infiltrated organs. Most notably, liver metastasis was completely abolished. As shown in Fig. 1, liver weight and cell proliferation levels in Ab-treated mice were significantly lower than those in untreated tumor-bearing mice (p < 0.0005) and were not significantly different from liver weight and cell proliferation levels in normal mice (p > 0.1), suggesting that the Abs inhibited tumor dissemination. However, since anti-Id monospecific Abs can destroy lymphoma cells and hence diminish tumor burden (15, 22), it could be argued that the anti-Id x anti-LFA-1 bispecific Abs act similarly to anti-Id Abs and have no added value as inhibitors of tumor spread. Therefore, we inoculated mice s.c. on day 0 with 38C-13 cells, treated them on day 2 and every 7 days thereafter with anti-Id or with anti-Id x anti-LFA-1 Abs, and sacrificed them on day 24. Because untreated mice develop tumors faster than Ab-treated animals (see below), mice in the untreated control group were inoculated with tumor on day 6 and sacrificed on day 24 (i.e., 18 days following inoculation). In this way mice in all experimental groups were sacrificed and assessed for tumor spread when their primary tumors were of the same mean size (602 ± 35 mm2 for untreated mice, 602 ± 52 mm2 for mice treated with anti-Id mAb, and 599 ± 38 mm2 for mice treated with bispecific mAb). The finding that primary tumors in mice treated with anti-Id or bispecific Abs reach a given size within a similar time, which is longer than that required in untreated mice, demonstrates that anti-Id and bispecific Abs have a similar attenuating effect on tumor growth. As shown in Fig. 2A, livers of tumor-bearing mice, treated with bispecific Abs, were devoid of proliferating cells, whereas livers of untreated mice or anti-Id-treated mice revealed high levels of cell proliferation. Hence, in contrast to anti-Id monospecific Abs, anti-Id x anti-LFA-1 bispecific Abs block liver metastasis. The superiority of the bispecific Abs compared with the anti-Id Abs cannot be accounted for by an insufficient dose of the anti-Id. Earlier studies in the 38C-13 tumor model indicated that an exceptionally low amount of anti-Id Abs mediates protection from tumor challenge (15, 16). Thus, one injection of 10 µg of the 2B6 anti-Id mAb mediated the maximal attainable tumor protection (15). Therefore, the anti-Id dosage used in the current study (several injections of 200 µg) was in excess of the maximal effective dose. It was chosen to equal the optimal dose of the bispecific Ab, which was determined in a dose-response study (not shown).



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  		FIGURE 1. Anti-Id x anti-LFA-1 bispecific Abs inhibit liver metastasis. Mice were inoculated s.c. with 1 x 105 38C-13 tumor cells, and injected i.p. with bispecific Abs (0.2 mg/mouse) 2 days later and every 7 days thereafter. Liver metastasis was determined by cell proliferation (A) and liver weight (B). The bars represent the mean values of 10 mice in each experimental group.

 


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  		FIGURE 2. Anti-Id x anti-LFA-1 bispecific Abs are superior to anti-Id monospecific Abs as inhibitors of liver and lymph node metastasis. Mice destined for Ab treatment were inoculated s.c. with 1 x 105 38C-13 cells on day 0 and injected i.p. with anti-Id or bispecific Abs (0.2 mg/mouse) on day 2 and every 7 days thereafter. Mice in the untreated experimental group were inoculated with tumor on day 6. All mice were sacrificed on day 24, and metastasis was determined by cell proliferation in liver (A), lymph nodes (B), and spleen (C). The bars represent the mean values of 10 mice in each experimental group.

 
The bispecific Abs also inhibited lymph node metastasis, albeit to a lesser degree than that observed in livers (Fig. 2B). Only distant lymph nodes were examined. Thus, when tumor cells were injected s.c. in one side of the animal, only lymph nodes from the opposite noninjected side were examined. These results demonstrate that while anti-Id monospecific Abs and anti-Id x anti-LFA-1 bispecific Abs mediate comparable attenuating effects on tumor growth, the bispecific Abs can additionally block tumor metastasis in lymph nodes and liver. The latter effects of bispecific Abs were organ specific. Thus, spleens of anti-Id-treated and anti-Id x anti-LFA-1-treated mice showed reduced tumor load compared with spleens of untreated mice, but the bispecific Abs were not superior to anti-Id Abs (Fig. 2C).

It should be noted that a variety of experimental designs were tested in which tumor was inoculated i.p. or s.c., and Abs were administered i.p. or i.v. The different routes yielded similar results. We prefer the s.c. route of tumor inoculation because the size of the primary s.c. tumors can be easily measured by caliper. The i.p. route of Ab administration is preferred because of its convenience. It should also be noted that the proliferation assays were performed on cell fractions isolated from liver by density gradient centrifugation. These cell fractions contained malignant and normal lymphoid cells, but no hepatocytes, and consisted mainly of malignant cells in metastatic livers and normal lymphocytes in nonmetastatic livers. Since we plated a total of 1 x 105 cells in each microplate well, the thymidine uptake rates reflect the proportion of proliferating cells in a sample of 1 x 105 isolated cells. The yield of fractionated cells was higher in metastatic livers compared with nonmetastatic livers (not shown). Therefore, the overall proliferation rates per organ are much higher in metastatic compared with nonmetastatic organs.

To verify tumor presence or absence in affected organs, spleen and liver cells of untreated and treated tumor-bearing mice were analyzed by flow cytometry for the expression of 38C-13 Id. As described above for the proliferation assays, fluorescence staining was performed on fractionated lymphoid cells (malignant and normal). The results of this analysis were in agreement with those of the proliferation assay. Whereas all the liver-derived cells of untreated tumor-bearing mice were Id-positive and hence represented tumor cells, livers of bispecific Ab-treated mice were devoid of tumor (Fig. 3). In contrast to the complete abolishment of liver metastasis, the effect of bispecific Abs on spleen was less dramatic, resulting in some decrease in splenic tumor mass. To ascertain that the unstained cells in livers and spleens of treated animals did not represent Id-negative tumor cells, we plated liver- and spleen-derived cells and followed them in cell cultures. While liver and spleen cell cultures of untreated mice yielded massive growth of tumor, no cells could be grown from livers of treated animals. Spleens of treated animals yielded massive growth of cells, which were all Id-positive (not shown), indicating that Ab treatment did not select for Id-negative tumor cells.



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  		FIGURE 3. Livers of tumor-bearing mice, treated with bispecific Abs, are devoid of Id-expressing cells. Mice were inoculated with tumor and treated as described in Fig. 2. Liver and spleen cells of untreated and bispecific Ab-treated mice were stained with mAb against 38C-13 Id (filled histograms) or with control Ab (open histograms).

 
We further assessed the bispecific Ab for its effect on the survival of tumor-bearing mice. As shown in Fig. 4, the survival rate of mice treated with the bispecific Ab is higher than the survival rate of mice treated with the 2B6 anti-Id Ab. Although this increase in survival rate was statistically nonsignificant, it was reproducible. It should be noted that Ab treatment was terminated on day 24. Preliminary results of our current studies reveal that continuation of treatment beyond day 24 (Ab injection every 7 days) further improves the survival rate of mice treated with bispecific Abs compared with mice treated with anti-Id Abs.



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  		FIGURE 4. Survival of bispecific Ab-treated animals. Mice (10/group) were inoculated s.c. with 1 x 105 38C-13 cells and injected i.p. with anti-Id or bispecific Abs (0.2 mg/mouse) 2 days later and every 7 days thereafter. Ab treatment was terminated on day 24.

 
The bispecific Ab contains two different classes of rat heavy chains: IgG1, originating from the 2B6 anti-Id mAb, and IgG2a, originating from the M17 anti-LFA-1 mAb. It could therefore be argued that the effect of the bispecific Ab may in essence result from a class switch of the anti-Id mAb to a more therapeutically effective class. However, similarly to previous findings in the murine model of 38C-13 (23) and in clinical trials (22), we demonstrated that the anti-Id response does not correlate with the subclass of the Ab. For this purpose we compared the therapeutic efficacy of a large panel of IgG1 and IgG2a rat anti-Id mAb that have comparable avidities and that cross-block each other. The 2B6 (IgG1) and D5D10 (IgG2a) mAb are representatives of this Ab panel. Fig. 5A shows that they have a similar effect on the growth of 38C-13 tumor. Moreover, similarly to our previous report on polyclonal goat anti-Id Abs (24), we demonstrate here that F(ab')2 of 2B6 are as efficient as the intact Ab in retardation of tumor growth (Fig. 5B). Hence, it is unlikely that the bispecific Ab is more effective than the anti-Id due to the difference in their Fc fragment.



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  		FIGURE 5. The therapeutic activity of anti-Id Abs does not depend on their isotype or the presence of Fc fragments. A, 2B6 (IgG1) and D5D10 (IgG2a) anti-Id mAb mediate a similar effect on survival of 38C-13 tumor bearing animals. Mice (10/group) were inoculated i.p. with 1 x 104 38C-13 cells and injected i.p. with mAb (50 µg/mouse) 2 days later. B, Intact molecules and F(ab')2 of 2B6 mAb mediate a similar effect on survival of 38C-13 tumor-bearing animals. Mice (10/group) were inoculated i.p. with 1 x 104 38C-13 cells, and injected i.p. with 2B6 mAb or its F(ab')2 (50 µg/mouse) 2 days later.

 
Anti-Id x anti-LFA-1 bispecific Abs inhibit migration of 38C-13 in an organ-specific manner

In view of the effects of the anti-Id and the bispecific Abs on tumor growth, it may be argued that the reduction in metastatic cells cannot be attributed to a reduction in metastasis per se despite the organ specificity associated with this reduction. We therefore studied the effect of the bispecific Ab on tumor cell trafficking. 51Cr-labeled cells were injected i.v., and their distribution in various organs was determined after 1 and 24 h. The results depicted in Fig. 6 demonstrate that the majority of cells left the blood circulation and migrated to the lung and liver at 1 h postinjection. Cell accumulation in the lungs was transient. Thus, by 24 h the cells had left the lungs, and their majority accumulated in the liver and spleen. This pattern of homing has been previously reported for tumor cells, including 38C-13C cells, as well as for activated T cells (25, 26, 27, 28). Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected (Fig. 6). In contrast, the anti-Id mAb had no effect on migration. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis results at least in part from reduced homing to these organs.



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  		FIGURE 6. Anti-Id x anti-LFA-1 bispecific Abs inhibit migration of 38C-13 cells to liver and lymph nodes. 51Cr-labeled 38C-13 cells (2 x 106; 8 x 105 cpm), mixed with 0.2 mg of anti-Id or bispecific Abs, were injected i.v. After 1 and 24 h, different organs were collected and assessed for radioactivity. The bars represent the mean values of five mice.

 
Anti-Id x anti-LFA-1 bispecific Abs do not affect DTH responses

Following the demonstration that the anti-Id x anti-LFA-1 bispecific Ab inhibited liver and lymph node metastasis, we demonstrated its specificity to tumor cells. Since anti-LFA-1 block immune responses, it was necessary to ensure that the bispecific Ab had no adverse effect on the immune system. It has been previously demonstrated that LFA-1/ICAM-1 interactions are indispensable for the generation of inflammatory responses, such as DTH, and that Abs against LFA-1 block these immune responses (9, 29). We therefore investigated the effect of the bispecific Ab on contact sensitivity to DNFB. The results are depicted in Fig. 7. In contrast to anti-LFA-1 Abs, which inhibited the response to DNFB, the bispecific Ab had no effect on the immune response. Hence, the anti-Id x anti-LFA-1 bispecific Ab could selectively block LFA-1-mediated tumor metastasis, leaving the immune response unaffected.



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  		FIGURE 7. Anti-Id x anti-LFA-1 bispecific Abs, in contrast to anti-LFA-1 monospecific Abs, do not inhibit DTH responses. Mice were sensitized with DNFB. Three days following sensitization, mice were injected i.p. with 0.2 mg of anti-LFA-1 mAb or bispecific Abs. Two days later, mice were challenged with hapten. Ear thickness was measured before challenge ({blacksquare}) and 24 h after challenge ({square}). The bars represent the mean values of five mice in each experimental group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate that bispecific Abs, directed against the adhesion molecule LFA-1 and against the Id of a B cell lymphoma, block tumor metastasis without affecting immune responses. Bispecific Abs with specificity to a tumor-specific Ag and to CD3 or CD16 have been previously applied for retargeting of cytotoxic effector cells to tumor cells (30, 31). Here we describe the application of a novel type of bispecific Abs, which selectively target tumor cells and prevent their metastatic spread. These Abs bind simultaneously to an adhesion receptor and to a tumor-specific Ag of an individual tumor cell. We have demonstrated that such bispecific Abs bind preferentially to tumor cells that coexpress both target Ags (14). It should be noted that the affinity of the parental Ab against the adhesion molecule should be lower than the affinity of the parental Ab against the tumor-specific Ag. Thus, the contribution of the antiadhesion molecule site of the bispecific Ab to its avidity toward the tumor should be minimal. A relatively low affinity of the antiadhesion Ab combined with high affinity of the Ab to the tumor Ag ensure that the bispecific Ab binds selectively to tumor cells, but not to nonmalignant cells.

It has been previously reported that LFA-1 is involved in liver metastasis of lymphoma, and that Abs to LFA-1 inhibit tumor metastasis (7, 8, 32). Our preliminary studies indicated that treatment of tumor-bearing mice with anti-LFA-1 mAb resulted in inhibition of tumor spread to the liver. However, Ab-treated mice showed accelerated growth of the primary tumor compared with untreated tumor-bearing mice (our unpublished observations). A possible explanation for the accelerated tumor growth in treated mice was that following binding to nonmalignant leukocytes, anti-LFA-1 Abs blocked immune responses, including those regulating host resistance to the tumor. We therefore proposed to target the anti-LFA-1 Abs to tumor cells by constructing a bispecific Ab directed against LFA-1 and against the tumor-specific Id. Indeed, this bispecific Ab blocked metastasis without affecting DTH responses. In addition, treatment with the bispecific Ab prolonged the survival of tumor-bearing mice.

Since the bispecific Ab contains two different classes of rat heavy chains, IgG1 and IgG2a, it could be argued that the effect of the bispecific Ab may result from a class switch of the IgG1 anti-Id mAb to a more therapeutically effective class. A study with class switch variants of murine anti-Id mAb indicated that IgG2a Abs had greater antitumor effects than IgG1 and IgG2b (33). However, more recent studies contradict this finding. Clinical trials revealed that major clinical responses were induced by Abs of the IgG1, IgG2a, or IgG2b subclass, and that the presence of tumor response or the magnitude of that response could not be correlated with the subclass of the Ab (22). In addition, Id vaccination studies in the 38C-13 model demonstrated that the IgG isotype of anti-Id Abs did not correlate with protective immunity (23). In agreement with these studies, our data indicate that IgG1 and IgG2a rat anti-Id mAb have a similar therapeutic effect in the 38C-13 model. Moreover, similar to our previous report on polyclonal goat anti-Id Abs (24), we demonstrate here that F(ab')2 of the anti-Id mAb 2B6 are as efficient as the intact Ab in the retardation of tumor growth. The mechanism of action of anti-Id Abs has not been elucidated. Since anti-Id have regulatory functions in the immune system (34, 35, 36), it has been suggested that inoculation of anti-Id Abs may have an antitumor effect by altering the Id-anti-Id network that plays a role in the regulation of B cell clones. Alternatively, It has been suggested that the anti-Id Abs exert direct antiproliferative effects, leading to programmed cell death (37, 38). In any case, we demonstrate that the type of Fc fragment does not have a significant impact on the therapeutic effect of anti-Id, and hence, it is unlikely that the bispecific Ab is more effective than the anti-Id due to the difference in their Fc fragments.

The present results indicate that while both Abs retard tumor growth, the bispecific Abs are superior to anti-Id Abs because they additionally inhibit tumor spread. However, although the bispecific Abs demonstrate organ specificity in their antitumor effect, it may nevertheless be argued that the reduction in metastatic cells cannot be attributed to the inhibition of metastasis per se. We therefore studied the effect of the bispecific Ab on tumor cell migration. Our data for homing of 38C-13 cells are in agreement with previously published data on 38C-13 and resemble the homing pattern of other lymphoid tumors (25, 26, 27, 28). The Ab inhibition studies demonstrate that, unlike anti-Id Abs, the anti-Id x anti-LFA-1 bispecific Abs inhibited migration of 38C-13 cells in a way that correlated with their inhibitory effect on metastasis. Tumor cells, like activated lymphocytes, accumulate in a very high proportion within the lung during the first hour after injection, but leave this organ almost completely after 2–3 h (25, 26, 27, 28). Our results demonstrate that the transient trapping of 38C-13 cells in the lung was not affected by the bispecific Ab. The liver represents another organ in which a high proportion of tumor cells and activated lymphocytes accumulate. While homing to the lung was transient, the accumulation of 38C-13 in the liver was constant. This accumulation was significantly inhibited by the bispecific Ab, but not by the anti-Id Ab. Hamann and Thiele (25) reported that anti-LFA-1 Abs did not inhibit homing of YAC-1 cells to the liver. This discrepancy may be accounted for by the difference in the cell types, YAC-1 being a T cell tumor and 38C-13 being a B cell tumor. Thus, different cell types may use different mechanisms for homing. Despite the massive development of 38C-13 lymph node metastases, only a small proportion of the i.v. injected cells migrated to peripheral lymph nodes, suggesting that the entry of a small number of lymphoma cells into lymph nodes is sufficient for the development of metastases. It has been demonstrated by Jonas et al. (27) that 38C-13 cells, in contrast to resting lymphocytes, reach the lymph nodes by an L-selectin-independent mechanism. In agreement with their data, we have recently reported that L-selectin is not required for lymph node metastasis of 38C-13, as anti-L-selectin did not inhibit tumor dissemination to peripheral lymph nodes, and L-selectin-negative 38C-13 variant cells disseminated as efficiently as wild-type cells (21). Here we demonstrate that the bispecific Ab reduced the migration rate of 38C-13 cells into lymph nodes, which correlates with its inhibitory effect on lymph node metastasis. Treatment with the bispecific Ab had no effect on migration to the spleen or on spleen metastasis compared with treatment with anti-Id Ab. This finding is in agreement with previous reports that the absence of LFA-1 does not interfere with lymphocyte homing to the spleen (39, 40). The migration study presented here indicates that the bispecific Ab-mediated reduction in liver and lymph node metastasis results at least in part from reduced homing to these organs.

The fate of Ab-blocked 38C-13 cells that cannot enter their target organ is not clear. When the entry of cells into one or several organs is blocked by Ab, the corresponding portion of cells may appear in peripheral blood or may give rise to increased accumulation of cells in other organs. However, we were unable to show higher percentages of cells in blood or in organs of bispecific Ab-treated animals. In addition to the organs presented in Fig. 6, we have determined cell migration to mesenteric lymph nodes, thymus, kidney, and bones (data not shown). Although we cannot exclude the possibility that the Ab-blocked tumor cells redistributed to an as yet unidentified organ, it seems that accumulation of the cells in such a putative organ may be less harmful than accumulation in the liver, because mice treated with bispecific Abs revealed higher survival rates than anti-Id-treated mice. The increased survival rates were statistically nonsignificant, but reproducible. As mentioned above, the Ab treatment was terminated on day 24. Preliminary results of our current studies reveal that continuation of a weekly treatment beyond day 24 results in significant improvement of survival rates. In any case, despite the present demonstration that with regard to survival, the bispecific Ab had little added benefit relative to the anti-Id, its inhibitory effect on liver metastasis may be highly beneficial. Hence, protection against liver metastasis is potentially important and useful in many clinical settings.

Lymphoid cells use distinct adhesion molecules for extravasation into different organs. LFA-1 plays an important accessory role in lymphocyte homing to lymph nodes, which is partially inhibited by anti-LFA-1 Abs. In contrast, LFA-1 does not play an important role in lymphocyte migration to the spleen (25, 39, 40). Similarly, it has been suggested that interactions between tumor cells and endothelium at the site of extravasation represent one mechanism that determines organ-specific metastasis. Although the homing behavior of lymphoma cells closely resembles that of activated lymphocytes (25), the role of LFA-1 in migration of the two cell types may differ. Hence, while lymphocytes and lymphoma cells similarly require LFA-1 for lymph node homing, but not for spleen homing, they differ in the mechanisms regulating migration to the liver. While LFA-1 is involved in liver metastasis of lymphoma (7, 8, 32), it does not play a role in lymphocyte homing to the liver (25). The organ specificity of adhesion molecules may account for our finding that the bispecific Ab fully abolished liver metastasis, but only partially inhibited metastasis in other organs. In addition, tumor cells may use more than one adhesion receptor to mediate similar metastatic activities. Such a redundancy may make it difficult to ascribe a particular activity to a single receptor and to block dissemination to a specific organ by a single Ab. Hence, although inhibition of one adhesion receptor may not affect tumor spreading to a particular organ, simultaneous blockade of a set of adhesion molecules may be required for complete inhibition of tumor dissemination. Administration of a cocktail of bispecific Abs, directed against Id and several adhesion molecules, may therefore be required.

The present results suggest that bispecific Ab treatment when applied together with standard therapy may selectively block tumor cell dissemination and hence prevent tumor spread beyond its level at diagnosis. The present experimental system of lymphoma may serve as a model for different solid tumors, where patients who present with localized tumors may benefit from the combination of established therapies with selective targeting of tumor cells for prevention of their spread.


   Footnotes
 
1 This work was supported by grants from the Chief Scientist’s Office of the Israel Ministry of Health, the Israel Cancer Association, the Cancer Biology Research Center of Tel Aviv University, the Karl and Leonora Fingerhut Fund for Cancer Research, and the Research Fund of Tel Aviv University. Back

2 Address correspondence and reprint requests to Dr. Nurit Hollander, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail address: hollandn{at}post.tau.ac.il Back

3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; DNFB, 2,4-dinitro-1-fluorobenzene. Back

Received for publication October 16, 2002. Accepted for publication December 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. Avin, J. Haimovich, and N. Hollander
Anti-Idiotype x Anti-CD44 Bispecific Antibodies Inhibit Invasion of Lymphoid Organs by B Cell Lymphoma
J. Immunol., October 1, 2004; 173(7): 4736 - 4743.
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