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The Journal of Immunology, 2004, 173: 4736-4743.
Copyright © 2004 by The American Association of Immunologists

Anti-Idiotype x Anti-CD44 Bispecific Antibodies Inhibit Invasion of Lymphoid Organs by B Cell Lymphoma1

Esther Avin, 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
 
The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. 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 bifunctional 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. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id x anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id x anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphoid neoplasms display a wide spectrum of growth pattern, from limited spread to wide dissemination. Because normal lymphocytes circulate continuously and extravasate into lymphoid organs and into 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 inhibit lymphoma metastasis in experimental models (5, 6, 7, 8), emphasizing the clinical potential of such Abs. However, because nonmalignant cells express a variety of adhesion molecules as well, treatment with Abs against adhesion molecules may also block normal functions (9, 10, 11).

To circumvent adverse effects of anti-adhesion 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. This proposition was based on previous reports that bispecific Abs bind more avidly to cells that coexpress both target Ags than to cells that express only one target Ag (12, 13). Accordingly, bispecific Abs against an adhesion molecule and a tumor specific Ag may bind preferentially to tumor cells and inhibit their adhesion in a selective manner. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. 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, which is commonly used by us and by 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 blocked LFA-1-mediated adhesion of Id-expressing tumor cells, but had no effect on adhesion of normal lymphocytes (14). Moreover, it abolished liver metastasis in tumor-bearing mice (19). However, despite its dramatic inhibitory effect on liver metastasis, the anti-Id x anti-LFA-1 Ab had only partial effect on dissemination to lymph nodes, and no effect on spleen and bone marrow metastasis. The organ specificity of adhesion molecules may account for this selective effect of Abs and may suggest the need for simultaneous blockade of a set of adhesion molecules.

The adhesion molecule, CD44, and its ligand, hyaluronan, play a role in tumor growth, invasion, and metastasis (5, 20, 21, 22, 23, 24, 25, 26). Therefore, we produced anti-Id x anti-CD44 bispecific Abs. We demonstrate here that these bispecific Abs inhibited tumor dissemination to the lymph nodes, bone marrow, and spleen in mice carrying primary s.c. tumors. In contrast to anti-CD44 Abs, the bispecific Abs had no inhibitory effect on delayed-type hypersensitivity (DTH)3 responses. Hence, anti-Id x anti-CD44 bispecific Abs 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. The Tel Aviv University animal care and use committee approved all of the mice experiments.

Cell lines and Abs

38C-13, a carcinogen-induced B cell lymphoma, was maintained as described (27). DB2 is an Id-negative variant of the 38C-13 cell line (28). The hybridoma cell lines, 2B6 and S4C8, secrete rat (2B6) or mouse (S4C8) mAbs that are specific for an idiotypic determinant of the 38C-13 IgM (29). The 2B6 and S4C8 mAbs were purified by affinity chromatography on Id conjugated to Sepharose. The hybridoma cell lines, KM201 and KM81, secreting rat mAbs against murine CD44, were obtained from the American Type Culture Collection (Manassas, VA). The KM201 and KM81 mAbs were purified by protein G-Sepharose fractionation. The anti-Id x anti-CD44 bispecific Abs were generated by somatic cell hybridization of the 2B6 or S4C8 cells with the KM201 cells as previously described for the anti-Id x anti-LFA-1 bispecific Ab (14). They were purified by affinity chromatography on a column of Id coupled to Sepharose. The Id column removed the monospecific anti-CD44 Abs while retaining the monospecific anti-Id Abs and the bispecific Abs. The adsorbed Abs were eluted by a pH gradient as previously described (14). For the 4H8 bispecific Ab, the fraction containing the bispecific Ab peaked at pH 4.5, while the fraction containing the anti-Id monospecific Ab 2B6 was eluted at pH 3.0–3.5. For the A1/4 bispecific Ab, the fraction containing the bispecific Ab peaked at pH 5.2, while the anti-Id monospecific Ab S4C8 was eluted at pH 4.5–4.9. The purified fractions were analyzed by SDS-PAGE and ELISA as described (14). The yield was 0.6 mg/ml ascites for the 4H8 Ab, and 1.2 mg/ml ascites for the A1/4 Ab.

Tumor inoculation and Ab treatment

Mice were inoculated s.c. with 1 x 105 38C-13 cells. Abs (0.2 mg) were injected i.p. 2 days later and every 7 days thereafter.

Determination of tumor metastasis

Tumor dissemination was determined by cell proliferation assays as described (30). Briefly, lymph node, spleen, and bone marrow single-cell suspensions were incubated for 24 h at 1 x 105 cells per well in 96-well flat-bottom microplates. They were then pulsed for 20 h with 1 µCi/well of [methyl-3H]thymidine (Amersham Biosciences, Buckinghamshire, U.K.) and harvested. Thymidine uptake was assessed by liquid scintillation counting. Under these conditions, the presence of 38C-13 cells in the lymphoid organs 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.

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. The 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 20 h, mice were sacrificed, and different organs were collected. The radioactivity of whole organs was counted in a gamma counter. Data are presented as percent of input counts in whole organs.

Sensitization and elicitation of DTH

Twenty microliters of 1% 2,4-dinitro-1-fluorobenzene (DNFB; Sigma-Aldrich, St. Louis, MO) in 4/1 acetone/olive oil (Sigma-Aldrich, St. Louis, MO) 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.

Adhesion assay

Tumor cells were incubated overnight with 1µCi/ml [methyl-3H]thymidine (Amersham Biosciences). After washing, 5 x 104 radio-labeled cells were incubated for 1 h at 37°C in flat-bottom 96-well microplates that had been precoated overnight at 4°C with 0.5 mg/ml hyaluronic acid (HA; Sigma-Aldrich) and then blocked with PBS containing 1% BSA for 1 h at 37°C. Following the incubation of cells in HA-coated microplates, the wells were washed extensively to remove nonadherent cells. Adherent cells were solubilized in 150 µl of 1 N NaOH, and radioactivity was determined by a liquid scintillation counter.

Flow cytometry

Cells were incubated for 10 min at 4°C with 60 µg of aggregated human IgG (Sigma-Aldrich) to block FcR. Primary Abs were then added to cells and incubated for 45 min at 4°C. The cells were washed in PBS containing 0.5% BSA and incubated for 45 min at 4°C with FITC-conjugated F(ab')2 of goat anti-mouse IgG or mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were washed and analyzed by a FACSort flow cytometer (BD Biosciences, Mountain View, CA).

ELISA

Bispecific Abs were analyzed by ELISA. Id protein at a concentration of 10 µg/ml was adsorbed to 96-well plates for 18 h at 4°C. Plates were then washed, blocked with 1% BSA for 1 h at 37°C, and washed. Serial dilutions of samples were then added to plates for 2 h at room temperature. Plates were washed and HRP-conjugated rabbit anti-mouse IgG or HRP-conjugated mouse anti-rat IgG (Jackson ImmunoResearch Laboratories) were added for 1 h at room temperature. The plates were washed and developed using 1,2-orthophenylenediamine substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-Id x anti-CD44 bispecific Abs bind preferentially to Id-expressing tumor cells

We have generated two types of anti-Id x anti-CD44 bispecific Abs, which are directed against an idiotypic determinant of the murine B lymphocyte tumor 38C-13, and against the adhesion molecule CD44. The 4H8 Ab is a fusion product of rat anti-Id (IgG1) and rat anti-CD44 (IgG1), and therefore, consists entirely of rat IgG1. The A1/4 Ab is a fusion product of mouse anti-Id (IgG1) and rat anti-CD44 (IgG1), and therefore, consists of rat x mouse IgG1 hybrid. The anti-CD44 mAb KM201, which was used for fusion, binds to CD44H, the hemopoietic (standard) isoform of CD44. As discussed below, KM201 was selected because of its relatively low affinity compared with other anti-CD44 Abs, such as KM81 (Fig. 1).



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  		FIGURE 1. Preferential binding of anti-Id x anti-CD44 bispecific Abs to Id-positive cells. The bispecific Abs A1/4 (A) and 4H8 (B) were incubated at the indicated concentrations with Id-positive 38C-13 cells, with Id-negative DB2 cells, or with normal lymph node cells. Following washing, cells were stained with FITC-labeled secondary Abs. C, Staining of 38C-13 cells with 2B6 anti-Id (open gray histogram). D, Staining of 38C-13 cells with the anti-CD44 KM201 (shaded histogram) or KM81 (dotted histogram). E and F, Staining of the DB2 cells (E) and normal lymph node cells (F) with 2B6 anti-Id (open gray histograms) or with KM201 anti-CD44 (shaded histograms). The open black histograms in C–F depict control staining.

 
We tested Ab binding to cells that coexpress both target Ags, namely Id and CD44, in comparison to cells that express only CD44. We compared binding to the 38C-13 cell line with binding to its Id-negative variant DB2 (28). As shown in Fig. 1, although 38C-13 and DB2 cells express similar amounts of CD44, the bispecific Abs bind to 38C-13 cells more efficiently than to DB2 cells, which are Id-negative, and hence, express only one target Ag. A similar comparison of binding to 38C-13 cells with binding to lymph node cells from normal C3H/eB mice demonstrated that the bispecific Abs do not bind to normal lymphocytes (Fig. 1). Fig. 1 also demonstrates that the preferential binding to cells that coexpress the tumor-specific Ag and the adhesion molecule is common to the 4H8 (rat x rat) and the A1/4 (rat x mouse) bispecific Abs.

Because the bispecific Abs did not bind to Id-negative CD44-positive cells, their CD44 specificity could be questioned. Therefore, the specificity of these Abs was further verified by competition with the CD44 ligand, HA. It was demonstrated that, similarly to the monospecific anti-CD44 Ab KM201, the bispecific Ab inhibited binding of soluble HA to 38C-13 cells (Fig. 2). In contrast, the monospecific anti-Id Ab had no inhibiting effect. Thus, the bispecific Ab is directed against CD44.



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  		FIGURE 2. Anti-Id x anti-CD44 bispecific Abs inhibit binding of soluble HA to cells. The 38C-13 cells were incubated with 20 µg/ml FITC-labeled HA in the absence (A) or in the presence of 60 µg/ml unlabeled 2B6 anti-Id Ab (B), KM201 anti-CD44 Ab (C), or 4H8 bispecific Ab (D). Shaded histograms depict binding in the absence of Ab. Open histograms depict binding in the presence of Ab.

 
Anti-Id x anti-CD44 bispecific Abs inhibit adhesion of Id-expressing tumor cells

The bispecific Abs were tested for their effect on cell adhesion to immobilized HA. To verify that cell adhesion in our assays resulted from specific recognition of HA and not from nonspecific sticking, we demonstrated that cell adhesion to immobilized HA was inhibited by addition of soluble HA to the culture medium (Fig. 3A). Cell adhesion was also inhibited by the anti-CD44 Ab, KM201 (Fig. 3B), indicating that the adhesion was mediated by CD44 binding to its ligand, HA. The capacity of the anti-CD44 mAb KM201 to inhibit cell adhesion has been documented (31) and is confirmed in the present study (Fig. 3). In contrast to the monospecific anti-CD44 Ab, which inhibited adhesion of both 38C-13 cells and DB2 cells, the bispecific anti-Id x anti-CD44 affected only cells that express the Id. Thus, whereas adhesion of 38C-13 cells to hyaluronate was inhibited by the bispecific Abs, adhesion of DB2 cells was resistant to inhibition (Fig. 3). The two types of anti-Id x anti-CD44 bispecific Abs, 4H8 and A1/4, were equally effective in blocking 38C-13 cell adhesion. The monospecific anti-Id Ab had no effect on adhesion. These results indicate that the bispecific Abs can preferentially inhibit adhesion of cells that coexpress the tumor-specific Ag and CD44.



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  		FIGURE 3. Anti-Id x anti-CD44 bispecific Abs selectively block adhesion of Id-positive cells to immobilized HA. A, [3H]Thymidine-labeled 38C-13 cells were added in medium alone or along with 2 mg/ml soluble HA to HA-coated microplate wells. B, [3H]Thymidine-labeled 38C-13 or DB2 cells were added to HA-coated microplate wells along with 10 µg/ml anti-Id, anti-CD44, and the 4H8 and A1/4 bispecific Abs. The plates were incubated for 1 h, and the percentage of adherent cells was determined as described in Materials and Methods.

 
In vivo clearance of the bispecific Abs

To investigate the in vivo clearance from the serum, naive mice were injected with 0.2 mg of the bispecific Abs. The mice were bled at various time points, and the presence of Abs was determined by an ELISA. As shown in Fig. 4, measurable concentrations of bispecific Abs could be detected up to 10 days postinjection. The clearance kinetics of the bispecific Abs was comparable to that observed for the monospecific anti-Id Ab (data not shown). Although the clearance of the two bispecific Abs was essentially similar, it was somewhat slower for the A1/4 compared with the 4H8 bispecific Ab.



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  		FIGURE 4. In vivo clearance of anti-Id x anti-CD44 bispecific Abs. A total of 0.2 mg of 4H8 (A) and A1/4 (B) bispecific Abs were injected to naive mice. The animals were bled at various time points, and the presence of Abs was determined by an ELISA. Ab concentration in the samples of time point 0 (5 min postinjection) was taken as 100%.

 
Anti-Id x anti-CD44 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 (19, 29, 30). Tumor dissemination is manifested by enlargement of the infiltrated organs, as determined by an increase in organ weight. Presence of tumor cells in the different organs was determined by staining with tumor-specific anti-Id Abs. 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-CD44 bispecific Abs on tumor metastasis, 38C-13-inoculated mice were treated with 0.2 mg 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. In preliminary studies, the bispecific Abs attenuated the growth of the s.c. primary tumors, and reduced tumor load in lymph nodes, bone marrow, and spleen (data not shown). The anti-Id x anti-CD44 bispecific Abs attenuated the growth of the primary s.c. tumors, as compared with untreated tumor-bearing mice, because they can kill lymphoma cells similarly to anti-Id monospecific Abs (15, 32). Therefore, it could be argued that the reduced tumor burden in infiltrated organs resulted from a delay in the onset of metastasis from the attenuated primary tumor rather than from inhibition of homing. Therefore, mice were inoculated with tumor cells at different times. We inoculated mice s.c. on day 0 with 38C-13 cells, treated them with Abs on day 2 and every 7 days thereafter, and sacrificed them on day 24. Because untreated mice develop tumors faster than Ab-treated animals, mice of the untreated control group were inoculated with tumor cells on day 7, and sacrificed on day 24 (i.e., 17 days following inoculation). By this way, mice of all experimental groups were sacrificed and assessed for tumor spread when their primary tumors were of a similar mean size. 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. However, as shown in Fig. 5, treatment with bispecific Abs additionally inhibited tumor dissemination to peripheral lymph nodes, spleen, and bone marrow. 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. The superiority of the bispecific Abs as 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). 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 5. Anti-Id x anti-CD44 bispecific Abs inhibit 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 with A1/4 bispecific Abs (0.2 mg/mouse) on day 2 and every 7 days thereafter. Mice of the untreated experimental group were inoculated with tumor cells on day 7. All mice were sacrificed on day 24, and metastasis was determined by cell proliferation in the spleen (A), lymph nodes (B), and bone marrow (C). The bars represent mean values of 10 mice in each experimental group.

 
All together, these results demonstrate that while anti-Id monospecific Abs and anti-Id x anti-CD44 bispecific Abs mediate comparable attenuating effects on tumor growth, the bispecific Abs can additionally block tumor metastasis in lymph nodes, spleen, and bone marrow. The two types of bispecific Abs, 4H8 and A1/4, consistently mediated identical effects (data not shown). Because the yield of the purified A1/4 is higher than the yield of 4H8, we prefer to use the A1/4 Ab.

It should be noted that the proliferation assays were performed on cell suspensions isolated from the different lymphoid organs. These cell suspensions contained malignant and normal cells. Because 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 cells was higher in metastatic organs compared with nonmetastatic organs (not shown). Therefore, the overall proliferation rates per organ are even much higher in metastatic compared with nonmetastatic organs.

To verify tumor presence or absence in affected organs, lymph node and bone marrow cells of untreated and treated tumor-bearing mice were analyzed by flow cytometry for expression of 38C-13 Id. The results of this analysis were in agreement with those of the proliferation assay. Whereas lymph nodes and bone marrow of untreated tumor-bearing mice were loaded with a large number of Id-positive cells that represented tumor cells, lymph nodes and bone marrow of bispecific Ab-treated mice consisted mainly of Id-negative nonmalignant cells (Fig. 6). To ascertain that the unstained cells in lymph nodes and bone marrow of treated animals did not represent Id-negative tumor cells, we plated lymph node- and bone marrow-derived cells and followed them in cell cultures. Although cultures of untreated mice yielded massive tumor growth, few or no cells could be grown from the lymphoid organs of treated animals. The few cells that occasionally grew from organs of treated animals were all Id-positive (data not shown), indicating that Ab treatment did not select for Id-negative tumor cells.



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  		FIGURE 6. Treatment of tumor-bearing mice with bispecific Abs inhibits accumulation of Id-expressing cells in the lymph nodes and bone marrow. Mice were inoculated with tumor cells and treated with A1/4 bispecific Abs as described in legends to Fig. 5. Lymph nodes and bone marrow cells of untreated and bispecific Ab-treated mice were stained with mAb against 38C-13 Id (filled histograms) or with control mAb (open histograms).

 
Anti-Id x anti-CD44 bispecific Abs inhibit migration of 38C-13 to lymphoid organs

As discussed above, due to effects of the Abs on tumor growth, it could be argued that the reduction in metastatic cells may not be attributed to reduction in metastasis per se. Therefore, we studied the effect of the bispecific Abs on tumor cell trafficking. 51Cr-labeled cells were injected i.v., and their distribution in lymph nodes, bone marrow, and spleen was determined after 20 h. The results depicted in Fig. 7 demonstrate that migration of 38C-13 tumor cells to peripheral lymph nodes, bone marrow, and spleen was inhibited by the bispecific Abs as compared with the anti-Id Abs. Hence, the bispecific Ab-mediated reduction in lymph node, bone marrow, and spleen metastasis results, at least in part, from reduced homing to these organs.



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  		FIGURE 7. Anti-Id x anti-CD44 bispecific Abs inhibit migration of 38C-13 cells to lymphoid organs. 51Cr-labeled 38C-13 cells (2 x 106 cells; 9 x 105 cpm), mixed with 0.2 mg of anti-Id or bispecific Abs, were injected i.v. After 20 h, different organs were collected and assessed for their radioactivity. The bars represent mean values of five mice. Inhibition of migration by the bispecific Ab compared with the anti-Id Ab was statistically significant (p < 0.005 for peripheral lymph nodes, p < 0.05 for mesenteric lymph nodes, p < 0.005 for bone marrow, and p < 0.005 for the spleen).

 
Anti-Id x anti-CD44 bispecific Abs prolong survival of tumor-bearing mice

We further assessed the bispecific Abs for their effect on survival of tumor-bearing mice. As shown in Fig. 8, the survival rate of mice treated with the bispecific Abs is higher than the survival rate of mice treated with the anti-Id Abs. This increase in survival rate was statistically significant (p < 0.01). It should be noted that we intentionally inoculated 1 x 105 tumor cells, which is a very high dose for the highly tumorigenic 38C-13 tumor, and which is therefore, minimally affected by anti-Id Ab treatment.



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  		FIGURE 8. Anti-Id x anti-CD44 bispecific Abs prolong survival of tumor-bearing animals. Mice (10 per group) were inoculated s.c. with 1 x 105 38C-13 cells, and injected i.p. with anti-Id or with A1/4 bispecific Abs (0.2 mg per mouse) 2 days later and every 7 days thereafter. Ab treatment was terminated on day 23. Survival of the two Ab-treated groups was prolonged compared with the control group (p < 0.005). The bispecific Ab was more efficient than the anti-Id Ab (p < 0.01).

 
Anti-Id x anti-CD44 bispecific Abs do not affect DTH responses

Following the demonstration that anti-Id x anti-CD44 bispecific Abs inhibited metastasis, we demonstrated their in vivo specificity to tumor cells. Because anti-CD44 block immune responses, it was necessary to ensure that the bispecific Abs had no adverse effects on the immune system. It has been previously demonstrated that interactions of CD44 with its ligands are indispensable for the generation of inflammatory responses, such as DTH, and that Abs against CD44 block these immune responses (10, 33). Therefore, we investigated the effect of the bispecific Abs on contact sensitivity to DNFB. The results are depicted in Fig. 9. In contrast to anti-CD44 Abs, which inhibited the response to DNFB, the bispecific Abs had no effect on the immune response. Hence, the anti-Id x anti-CD44 bispecific Abs could selectively block CD44-mediated tumor metastasis, leaving the immune response unaffected.



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  		FIGURE 9. Anti-Id x anti-CD44 bispecific Abs, in contrast to anti-CD44 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-CD44 or bispecific Abs. Two days later, mice were challenged with hapten. Ear thickness was measured before the challenge (filled bars) and 24 h after the challenge (open bars). The bars represent mean values of five mice in each experimental group. The anti-CD44-treated animals showed a reduced ear swelling compared with the control animals (p < 0.005), whereas treatment with the bispecific Ab did not affect ear swelling compared with the controls (p < 0.25).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that bispecific Abs, directed against the adhesion molecule CD44 and against the Id of a B cell lymphoma, block tumor metastasis without affecting immune reactivity. 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 (34, 35). In this study, 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. It should be noted that we are using Ab combinations in which the affinity of the parental Ab against the adhesion molecule is lower than the affinity of the parental Ab against the tumor-specific Ag. Thus, the contribution of the anti-adhesion molecule site of the bispecific Ab to its avidity toward the tumor is minimal. A relatively low affinity of the anti-adhesion Ab, combined with high affinity of the Ab to the tumor Ag, ensures that the bispecific Ab binds selectively to tumor cells, but not to nonmalignant cells.

It has been previously reported that CD44 is involved in tumor growth, invasion, and metastasis (20, 21, 22, 23, 24, 25, 26). With regard to non-Hodgkin’s lymphoma, it has been shown that expression of CD44 correlates with the dissemination status of the tumor, and that CD44 negative lymphomas have a relatively good prognosis (36, 37, 38). Several reports have shown that inhibitors of CD44, including anti-CD44 mAbs, soluble CD44-Fc fusion proteins, and synthetic peptides can block tumor invasion and metastasis (5, 21, 22, 25, 39). However, treatment with such inhibitors may block as well normal leukocyte functions, including those, regulating host resistance to the tumor. Therefore, we proposed to target the anti-CD44 Abs to tumor cells by constructing bispecific Abs directed against CD44 and against the tumor-specific Id. Indeed, these bispecific Abs blocked metastasis without affecting DTH responses. In addition, treatment with the bispecific Abs prolonged survival of tumor-bearing mice. The bispecific Abs were constructed from the anti-pan-CD44 mAb KM201 directed against the constant region of CD44. Bispecific Abs consisting of anti-pan-CD44 may target the multiple isoforms of CD44, thus overriding the requirement for a specific anti-CD44 Ab for each isoform. Because the bispecific Abs bind selectively to cells that express a tumor specific Ag, there is no concern that they may bind to nonmalignant cells that express the standard form of CD44.

We used the hybrid hybridoma (quadroma) technique for production of bispecific Abs. We produced rat/rat and rat/mouse Abs for comparison. Conventional rat/rat or mouse/mouse hybrid hybridomas produce up to 10 different IgG molecules consisting of various H and L chain combinations (40, 41). Hence, the yield of functional bispecific Abs is very low. It has been reported that heavy chains preferentially associate with light chains of the same species (42). As a consequence, the incidence of the correctly paired Abs increases in rat/mouse hybrid hybridomas. Indeed, the yield of our anti-Id x anti-CD44 rat/mouse bispecific Ab was higher than the yield of the rat/rat Ab. However, with regard to their activity, there was no difference between the two Abs. The rat/rat and the rat/mouse bispecific Abs consistently mediated identical effects in all the functional tests.

The present results indicate that while both the anti-Id and the bispecific Abs retard tumor growth, the bispecific Abs are superior to anti-Id Abs because they additionally inhibit tumor spread. However, it may be argued that the reduction in metastasis cannot be attributed to inhibition of metastasis per se. Therefore, we studied the effect of the bispecific Abs on tumor cell migration. As previously reported by us (19) and by others (43), despite the massive development of 38C-13 lymph node metastases, only a small proportion of the i.v. injected cells migrated to peripheral and mesenteric lymph nodes, suggesting that the entry of a small number of lymphoma cells into lymph nodes is sufficient for development of metastases. It has been demonstrated by Jonas et al. (43) 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 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 (30). In this study, we demonstrate that the anti-Id x anti-CD44 bispecific Abs reduced the migration rate of 38C-13 cells into lymph nodes. The Abs also inhibited migration to bone marrow and partially inhibited migration to the spleen. The inhibitory effects of the bispecific Abs on homing correlated with their inhibitory effect on metastasis in these lymphoid organs. Altogether, the migration study presented here indicates that the bispecific Ab-mediated reduction in metastasis results, at least in part, from reduced homing to the lymphoid organs.

It should be emphasized that the reason for the bispecific Abs being more effective than the anti-Id is not due to a difference in their Fc fragment. In the first place, the 4H8 bispecific Ab is a fusion product of rat anti-Id (IgG1) and rat anti-CD44 (IgG1) and therefore consists entirely of rat IgG1, as is the case for the anti-Id mAb 2B6. In addition, we have previously demonstrated that F(ab')2 of the anti-Id mAb were as efficient as the intact Ab in retardation of tumor growth (19), indicating that the Fc fragment does not have a significant impact on the therapeutic effect of anti-Id.

Lymphoid cells use distinct adhesion molecules for extravasation into different organs. For instance, the integrin, LFA-1, plays an important accessory role in lymphocyte homing to lymph nodes, but not in lymphocyte migration to the spleen (44, 45, 46). 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 (44), it may differ from that of normal lymphoid cells. Hence, lymphocytes and lymphoma cells differ in the mechanisms regulating migration to the liver. Although LFA-1 is involved in liver metastasis of lymphoma (7, 8, 19, 47), it does not play a role in lymphocyte homing to the liver (44). The organ specificity of adhesion molecules may account for our previous finding that anti-Id x anti-LFA-1 bispecific Abs abolished liver metastasis, but had no significant effect on spleen metastasis (19). The present study demonstrates that anti-Id x anti-CD44 Abs inhibit spleen, lymph node, and bone marrow metastasis. The presently demonstrated inhibition of lymph node metastasis is in accordance with a previous demonstration that anti-CD44 Abs blocked invasion of lymph nodes by the murine T cell lymphoma LB (5). The 38C-13 and LB lymphoma cells probably express distinct CD44 molecules. Whereas LB cells are incapable of binding HA (48), 38C-13 cells can bind HA efficiently. This does not contradict the indispensable role of CD44 in lymphoma dissemination, as it has been demonstrated that there is no direct correlation between HA-binding and tumor spread (48). Thus, although HA is a principal ligand of CD44, other ligands may be involved in the CD44-dependent organ invasion. Mechanisms involved in spleen and bone marrow lymphocyte homing are complex and not fully understood. The {alpha}4{beta}1 (VLA-4) integrin has been shown to play a role in lymphocyte homing to spleen (49) and bone marrow (50). However, 38C-13 cells do not express membrane {alpha}4{beta}1 or {alpha}4{beta}7. Although they express the {alpha}4 subunit, they lack detectable protein or RNA transcripts for {beta}1 or {beta}7 (51). A similar phenomenon has been reported for other lymphoma cell lines. Thus, the T cell lymphoma, LB, expresses the {beta}1 and {beta}7 chains but lacks expression of the {alpha}4 chain, and therefore, does not express surface VLA-4 (52). Hence, members of the {beta}1 and {beta}7 integrin families are not involved in 38C-13 dissemination, which apparently depends on CD44. The predominant role of CD44 in metastasis of the 38C-13 lymphoma may correlate with the situation in man, where lymphomas that express low levels of CD44 are more often of stage I at diagnosis, disseminate less frequently, and have favorable prognosis, whereas patients with lymphomas having high levels of CD44 show tumor spread beyond stage II at diagnosis with unfavorable prognosis (36, 37, 38). The organ specificity of adhesion molecules may imply that the simultaneous blockade of a set of adhesion molecules may be required for complete inhibition of tumor dissemination. Administration of a mixture 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
 
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 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 Research Fund of Tel Aviv University, and the Karl and Leonora Fingerhut Fund for Cancer Research. This work was performed in partial fulfillment of the requirements for a Ph.D. degree of E.A. (Sackler Faculty of Medicine, Tel Aviv University, Israel). 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; HA, hyaluronic acid. Back

Received for publication February 5, 2004. Accepted for publication July 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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