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Originally published online as doi:10.1189/jlb.1105623 on April 7, 2006

Published online before print April 7, 2006
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(Journal of Leukocyte Biology. 2006;79:1157-1165.)
© 2006 by Society for Leukocyte Biology

Anti-CCR7 monoclonal antibodies as a novel tool for the treatment of chronic lymphocyte leukemia

Manuel Alfonso-Pérez*, Sonia López-Giral*, Nuria E. Quintana*, Javier Loscertales{dagger}, Patricia Martín-Jiménez{ddagger} and Cecilia Muñoz*,1

* Departments of Immunology and
{dagger} Hematology, Hospital Universitario de La Princesa, Madrid, Spain; and
{ddagger} Department of Hematology, Hospital Clínico Universitario de Salamanca, Spain

1Correspondence: Servicio de Inmunología, Hospital Universitario de La Princesa, C/Diego de León 62, 28006 Madrid, Spain. E-mail: cmunoz.hlpr{at}salud.madrid.org

ABSTRACT

To date, chronic lymphocytic leukemia (CLL) remains incurable with current treatments, which include the monoclonal antibodies (mAbs) rituximab and alemtuzumab. The efficacy of rituximab is modest when used as single agent, and alemtuzumab induces severe immunosuppression. To develop more potent and specific therapies, we propose the CC chemokine receptor 7 (CCR7) as an attractive target molecule to treat CLL, as it not only fulfills the requirements of a high-surface expression and a good level of tissue specificity, but it also plays a crucial role in mediating the migration of the tumor cells to lymph nodes (LNs) and thus, in the development of clinical lymphadenopathy. In the current work, murine anti-human CCR7 mAb mediated a potent, complement-dependent cytotoxicity (CDC) against CLL cells while sparing normal T lymphocytes from the same patients. The sensitivity to CDC was related to the antigenic density of CCR7. Moreover, these mAb blocked the in vitro migration of CLL cells in response to CC chemokine ligand 19 (CC219), one of the physiological ligands of CCR7. Conversely, CLL cells were poorly lysed through antibody-dependent, cell-mediated cytotoxicity (ADCC), probably as a result of the murine origin and the isotype of the anti-CCR7 mAb used. Molecular engineering techniques will allow us to obtain chimeric or humanized anti-CCR7 mAb to reach the best clinical response for this common and yet incurable leukemia.

Key Words: immunotherapy • chemokines • cytotoxicity • metastasis • chemotaxis • tumor

INTRODUCTION

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of tumor CD5+ B cells with a great resistance to undergo apoptosis. Despite its low proliferation index, peripheral blood lymphocyte count reaches high values, and leukemic cells demonstrate a marked tendency to invade lymph nodes (LNs), spleen, and bone marrow (BM).

The treatment of CLL is based on the use of purine analogs, particularly fludarabine, alone or in association, as a frontline regimen. To date, the only therapeutic combination resulting in a higher complete remission rate than the one obtained with fludarabine has been the use of rituximab, an anti-CD20 monoclonal antibody (mAb), in association with fludarabine or fludarabine plus cyclophosphamide [1 2 3 4 5 ]. Moreover, molecular remissions in BM aspirates have been achieved in CLL patients with the above combinations, raising the possibility that CLL may be potentially curable without stem cell transplantation. Obtaining the best initial response together with the elimination of CLL cells in the inoculum of the patients undergoing autologous transplantation constitute some of the main therapeutic challenges in CLL.

By virtue of the above considerations, the novel therapeutic strategies for CLL are now moving toward the use of more specific treatments, including mAb against different antigens expressed by CLL cells, which may hopefully cure this disease.

Antigen selection in immunotherapy must take into account the tumor specificity of the antigen, the antigenic density on the surface of the tumor cells, and the antigen modulation or internalization of the antigen-antibody complex, which can reduce the ability to produce cell death [6 ]. In most cases, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) are believed to be responsible for the clinical use of the unconjugated mAb, although the induction of apoptosis or cell-cycle arrest could also play a substantial role in other cases [7 , 8 ].

Among the possible targets for mAb therapy in CLL, we propose the CC chemokine receptor 7 (CCR7) as an interesting therapeutic target based on previous results from our group [9 ]: It is highly expressed in CLL cells, and tumor cells of patients presenting clinical lymphadenopathy exhibit a higher in vitro migratory response toward the ligands of CCR7, the homeostatic chemokines CC chemokine ligand 19 (CCL19; macrophage-inflammatory protein-3ß), and CCL21 (6Ckine) [9 , 10 ]. Therefore, blocking the entry of CLL cells into secondary lymphoid tissue with anti-CCR7 mAb could be crucial. In this sense, those hematological and nonhematological tumors expressing CCR7 have the ability to metastasize into secondary lymphoid organs, whereas tumors lacking this molecule or other chemokine receptors involved in the homing to secondary lymphoid organs present a minimal nodal dissemination [11 12 13 14 15 16 17 18 19 ]. Previously, CCR7 had been described to play a main role in the entry of lymphoid cells into the secondary lymphoid organs [20 21 22 23 24 25 ], including LNs, and its normal expression is restricted to naïve T and B lymphocytes and mature dendritic cells (DC) [26 27 28 29 30 31 32 ].

Therefore, we have tested the in vitro ability of two anti-CCR7 mAb to eliminate CLL cells, taking into account the high levels of functional CCR7 present on the surface of CLL cells as well as the lymphoid restriction of this molecule.

MATERIALS AND METHODS

Samples, reagents, and flow cytometry (FCM)
Peripheral blood samples from CLL patients and healthy donors were obtained after informed consent. An initial immunophenotypic characterization of whole blood cells was performed by standard four-color FCM with mAb directed against the following human surface antigens: CD45, CD19, {kappa}-light chain, {lambda}-light chain, CD20, CD23, CD5, and CD3 (all purchased from BD Biosciences, San Jose, CA). Samples with less than 60% of CLL cells on the mononuclear subpopulation were discarded. Analysis of CCR7, CD38, and {zeta}-associated protein-70 (ZAP-70) expression was subsequently performed on electronically gated tumor B cells or normal leukocyte subpopulations using phycoerythrin (PE)-conjugated mouse anti-human CCR7 (R&D Systems, McKinley Place, MN), PE-conjugated anti-human CD38 (BD Biosciences), and PE-conjugated anti-human ZAP-70 (Caltag, Burlingame, CA). In all cases, appropriate isotype controls (ICs) were included. Immunofluorescence staining was analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were isolated by ficoll gradient centrifugation (Histopaque-1077, Sigma-Aldrich, Madrid, Spain).

Macrophage-derived DC were obtained by in vitro differentiation of monocytes isolated from PBMC by plastic adherence and cultured for 7 days in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4. Then, DC were matured by culture with lipopolysaccharide for 24 h. Maturation was assessed by FCM analysis of appropriate surface markers.

Purified mouse anti-human CCR7 mAb were obtained from BD Biosciences [2H4 clone, immunoglobulin M (IgM) isotype] and R&D Systems (150503 clone, IgG2a isotype). Rituximab (Mabthera) was purchased from Roche Pharmaceuticals (Basel, Switzerland). DNA dye 7-aminoactinomycin D (7-AAD), used in apoptosis and cell viability assays, was purchased from BD Biosciences. Recombinant human chemokines CCL19 and CXC chemokine ligand 12 (CXCL12) were purchased from R&D Systems.

Ig heavy chain variable region (IgVH) mutation analysis
For amplification of complete VH diversity/joining (VDJH) rearrangements, six VH family-specific primers from the framework region 1 and one JH consensus primer were used in a multiplexed polymerase chain reaction (PCR) [33 ]. All reactions were carried out in 50 µl containing 0.1 µg DNA samples and 10 pmol each primer. The PCR conditions consisted of one cycle at 95°C for 10 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and one final cycle at 72°C for 30 min. PCR products were loaded on a 10% nondenaturing polyacrylamide gel in 1x Tris-acetate-EDTA buffer, run at room temperature, and visualized by ethidium bromide staining. PCR products were eluted from polyacrylamide gels and sequenced directly in an automated ABI 377 DNA sequencer using Big-Dye terminators [34 ] (Applied Biosystems, Foster City, CA). The VDJH sequences obtained were aligned with the human germ-line sequences presenting the highest homology from the V base [35 ], using on-line DNAPLOT (MRC Centre for Protein Engineering, Cambridge, UK). To confirm base changes in the germ-line IgH sequence, PCR amplifications were sequenced in the forward- and the reverse-amplified fragment to observe the same change in separate reactions. Sequences containing more than 2% deviation from the germ-line sequence were considered somatically mutated rather than genomic polymorphisms.

Receptor endocytosis assay
To study whether the anti-CCR7 mAb cause the internalization of their target chemokine receptor, 5 x 105 CLL cells were incubated with 2 µg/mL anti-CCR7 mAb for different periods of time (from 30 s to 1 h) at 37°C. CCL19, one of the physiological ligands of CCR7, which causes the endocytosis of the chemokine receptor, was used as a positive control. After the elimination of bound mAb or CCL19 by means of an acidic wash (0.1 M glycine, 0.15 M NaCl, pH=2.5), the expression of CCR7 was determined in CLL cells by FCM analysis as indicated above.

CDC
PBMC suspension (50 µL) containing 105 target cells was plated in a 96-well round-bottom plate together with the desired concentration of purified anti-CCR7 mAb or an IC. After 30 min of incubation at 37°C, the cells were centrifuged and washed. Then, baby rabbit complement (Serotec, Oxford, UK), diluted at the optimal concentration (25%) in RPMI-1640 medium, was added. After 1–2 h at 37°C, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 mAb and allophycocyanin-conjugated anti-CD5 mAb to discriminate between CLL cells and T cell populations and with 7-AAD as a viability exclusion dye. The percentage of nonviable cells was measured separately in each population, and the percentage of lysis with heat-inactivated complement was used to calculate the specific lysis with the formula: Specific lysis = 100 x (% dead cells with complement–% dead cells with inactivated complement)/(100–% dead cells with inactivated complement).

ADCC
Natural killer (NK) cells were isolated from PBMC obtained from buffy coats. A positive selection of the NK cells was performed by staining with PE-conjugated anti-CD56 mAb (BD Biosciences) followed by immunomagnetic separation with anti-PE-coupled micro-beads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). The purity of the sample, determined by FCM, ranged from 95% to 98%. Anti-CCR7-dependent cell-mediated cytotoxicity was measured by FCM after 4 h of incubation of CLL cells (target cells) with NK cells (as effector of cell lysis) at different effector-to-target (E:T) ratios in the presence or absence of the anti-CCR7 mAb or its IC. Then, the cells were stained with FITC-conjugated anti-CD19 mAb, PE-conjugated anti-CD56 mAb, and 7-AAD to discriminate CLL cells and effector NK cells and to analyze the viability of CLL cells, which when incubated without NK cells, were used as a control of spontaneous death, and the specific lysis was calculated as in the CDC assay.

Chemotaxis assay
CLL cells were treated when necessary with 2 µg/mL anti-CCR7 mAb for 30 min. Then, the chemotaxis of the same cells in response to CCL19 and to CXCL12, the ligand of CXC chemokine receptor 4 (CXCR4), was assayed in Transwell cell culture chambers (6.5 mm diameter, 10 µm thickness, 5 µm diameter pore size, Costar, Cambridge, MA). Briefly, 5 x 105 CLL cells, suspended in 100 µl RPMI-1640 medium with 0.5% bovine serum albumin (BSA), were added to the upper compartment of the chamber, and chemokines were added to the lower well in 600 µl of the same medium at the optimal concentration (1 µg/mL for CCL19 and 100 ng/mL for CXCL12). Migration was allowed to proceed for 4 h at 37°C in 5% CO2 atmosphere. Migrated cells were recovered from the lower chamber, stained with a FITC-conjugated anti-CD19 mAb, and counted by FCM for 60 s after calibrating the flow rate with Trucount tubes (BD Biosciences). Events were counted within the gated population of B cells and compared with the number of cells counted in the initial suspension of cells to calculate the percentage of input (100xnumber of cells migrated/number of cells counted in the initial suspension). Each experiment was performed in duplicate.

Apoptosis assay
In some experiments, 105 CLL cells were incubated with 2 µg/mL soluble anti-CCR7 mAb in 50 µL RPMI 1640 with 0.5% BSA. In others, anti-CCR7 mAb were attached to 96-well flat-bottom plates by overnight incubation in RPMI-1640 medium. Then, the plate was washed, and 105 CLL cells were added.

In both cases, the cells were incubated at 37°C for a maximum of 48 h. Cells were washed with cold phosphate-buffered saline (PBS), stained with FITC-conjugated anti-CD19 mAb, resuspended in 0.5 mL PBS, and diluted in 5 mL ice-cold 70% ethanol for overnight fixation at –20°C. The next day, the fixed cells were centrifuged, washed, and stained with 20 µg/mL 7-AAD for DNA content analysis. A minimum of 104 total events was acquired and analyzed using Cell-Quest Pro software (BD Biosciences). Briefly, CD19-positive events were gated, and doublets were discarded using pulse-area and pulse-width of the 7-AAD fluorescence. Percentages of sub-G1 peak corresponding to apoptotic cells were analyzed in the 7-AAD fluorescence histogram.

Proliferation assay
PBMC were isolated from healthy donors and cultured for 72 h in 96-well plates previously coated with anti-CCR7 mAb or their respective ICs. Positive controls such as anti-CD3 mAb plus IL-2 were also included. All measures were performed in triplicate. The cells were labeled with 1 µCi [3H]-thymidine (Amersham Biosciences GmbH, Freiburg, Germany) per well, along the last 16 h of the culture. Then, the cells were harvested and subjected to scintillation counting.

Statistical analysis
All statistical tests were performed in Statistical Package for the Social Sciences Version 8.0 (SPSS, Chicago, IL). In most assays, Wilcoxon or Kendall’s W nonparametric tests were used to compare the results obtained with the different treatments and to correlate clinical data and prognostic factors with CCR7 expression. Kruskal Wallis test was used for the study of differences in CCR7 expression among the different Rai stages. ANOVA was used for comparison between groups in [3H]-thymidine assay. Pearson correlation coefficient was calculated to study the association between CCR7 mean fluorescence intensity (MFI) and percentage of lysis in CDC.

RESULTS

Anti-CCR7 mAb do not induce internalization of their target
The use of therapeutical mAb in their unconjugated form requires not only a high density of the target antigen but also the presence of antigen-antibody complexes on the surface of the cells to activate complement or bind Fc receptors (FcRs) of cytotoxic cells; i.e., it is important that the binding of the mAb to the antigen does not induce its internalization, as this could decrease the therapeutic effect of the mAb. In this regard, it has been suggested that antibody binding to some chemokine receptors could result in the endocytosis of the receptor followed by its proteolysis or re-expression after antibody degradation [36 , 37 ]. Therefore, we analyzed if the binding of the mAb to CCR7 induced its internalization in CLL cells.

As we demonstrated in a previous work [9 ], the expression of CCR7 on the surface of CLL cells was two- to fourfold higher than in normal B or T cells or mature DC (Fig. 1A and 1B ) and did not decrease after the incubation of these cells with the anti-CCR7 mAb for different times up to 60 min (Fig. 1C) . Conversely, it decreased ~60% in the first 5 min of incubation with 1 µg/mL CCL19, which served as positive control.


Figure 1
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  		Figure 1. CCR7 surface expression is greater in tumor CLL cells than in normal B and T lymphocytes and does not decrease after binding of anti-CCR7 mAb. Peripheral blood samples from CLL patients (n=11), healthy donors (n=6), and macrophage-derived mature DC (n=6) were analyzed by FCM to discriminate different lymphoid populations and their corresponding CCR7 surface density measured as MFI. (A) Bars represent mean ± SD of CCR7 MFI. (B) A representative case is shown. (C) PBMC from CLL patients were incubated with the anti-CCR7 mAb (2 µg/mL) or CCL19 (1 µg/mL) for different periods of time between 30 s and 60 min. Then, CCR7 MFI was determined by FCM in electronically gated CD19+CD5+ CLL cells. Lines represent the expression of CCR7 relative to the basal CCR7 MFI. A representative case is shown.

 
Anti-CCR7 mAb mediate strong and specific in vitro CDC of CLL cells
The cytotoxicity mediated by the recruitment of complement proteins (CDC) or effector cells such as NK cells (ADCC) is the main mechanism of action proposed for the in vivo elimination of tumor cells by unconjugated, therapeutic mAb. Therefore, the potency of the anti-CCR7 mAb in mediating CDC was tested with different CLL samples. As shown in Figure 2A , a high proportion of CLL cells preincubated with the anti-CCR7 IgM mAb was killed after 1 h of treatment with rabbit complement. Similarly, the anti-CCR7 mAb with the IgG isotype also mediated a significant CDC (Fig. 2B) , however, lower than the IgM. This difference reflects the ability of IgM antibodies to activate the complement cascade. No significant CDC was observed when irrelevant Igs of the same isotype were used (Fig. 2A and 2B) . Dose-response experiments showed that concentrations as low as 1 µg/mL of both anti-CCR7 mAb were sufficient to mediate CDC of CLL cells (Fig. 2C and 2D) .


Figure 2
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  		Figure 2. Both anti-CCR7 mAb mediate strong and specific CDC of CLL cells. (A and B) PBMC from CLL patients (n=11) and macrophage-derived, mature DC from healthy donors (n=6) were incubated with anti-CCR7 mAb (2 µg/mL) or their respective ICs and then exposed to rabbit complement for 1 h. Cell lysis was determined by 7-AAD incorporation and FCM analysis in electronically gated CLL cells and normal T cells. Bars represent mean ± SD. (C and D) CDC assays with PBMC from CLL patients (n=5) and healthy donors (n=2) were performed with concentrations of the anti-CCR7 mAb from 0.5 to 16 µg/mL. Percentage of cell lysis as a result of CDC was determined by 7-AAD incorporation and FCM analysis in electronically gated CLL cells, T lymphocytes from CLL patients, and normal B and T cells from healthy donors. Lines represent mean of cell lysis.

 
Despite the comparatively lower efficacy of the anti-CCR7 mAb of the IgG isotype in causing CDC, kinetic assays demonstrated that its complement-activating capacity can be increased significantly by extending the time of incubation with the complement source (data not shown). This may be a result of a slower activation of complement cascade by mAb with IgG2a isotype.

CCR7 is not restricted to CLL cells, being also expressed by certain normal lymphoid subpopulations, mainly naïve B and T lymphocytes, and mature DC, which could be susceptible to cytotoxicity mediated by anti-C CR7 mAbs. It is interesting that CDC mediated by anti-CCR7 mAb was significantly higher (P=0.001 and P=0.016 for IgM and IgG mAb, respectively) in CLL cells than in T cells from the same patients. Similarly, B and T lymphocytes (Fig. 2C and 2D) and mature DC (Fig. 2A and 2B) from healthy donors were much more resistant to complement activity when treated under the same experimental conditions as above and even with higher doses of both anti-CCR7 mAb, as demonstrated in dose-response assays.

A possible explanation for the different sensitivity to CDC activity between normal leukocytes and CLL cells is the much lower expression of CCR7 in the former (Fig. 1A and 1B) , which in addition, is partially negative for CCR7. Indeed, the levels of expression of CCR7 in CLL cells significantly (r=0.602, P=0.025, n=11) correlated with the sensitivity to CDC of the different samples (Fig. 3 ), confirming the importance of the antigen density in the selection of a target for immunotherapy. Such differences in CCR7 expression did not correlate with the stages of the disease in a larger group of 45 CLL patients (Table 1 ). Similarly we found no significant correlation between CDC sensitivity and other clinical and prognostic factors of the patients, summarized in Table 2 .


Figure 3
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  		Figure 3. CDC potency correlates with CCR7 expression levels. CCR7 surface density of CLL cells correlated (r=0.602, P=0.025, n=11) with the percentage of lysis as a result of CDC mediated by 2 µg/mL of the anti-CCR7 IgM mAb after 1 h of incubation with rabbit complement.

 

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  		Table 1. Statistical Descriptives of CCR7 MFI of 45 Samples Grouped by Rai Stage

 

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  		Table 2. Clinical Data of the Patients

 
Moreover, in concordance with other works [38 ], differences in CDC activity did not seem to be related to a differential expression of complement regulatory proteins such as CD55 or CD59, as their levels of expression were similar among different CLL samples and between CLL cells and T lymphocytes from these patients (data not shown).

In vitro ADCC of CLL cells
The other main mechanism mediating the beneficial effects of the therapeutic mAb is the ADCC. Therefore, we tested the ability of human NK cells to lyse CLL cells previously treated with the anti-CCR7 IgG mAb. In six experiments performed with E:T ratios between 5:1 and 40:1, no significant ADCC was found in the presence of this mAb (mean lysis=13%±1, n=6) when compared with an irrelevant Ig with the same isotype (mean lysis=12%±5, n=6; Fig. 4 ). This is probably a result of the low affinity of the human FcRs for the murine mAb or the low activity of the murine IgG2a isotype in ADCC. Conversely, rituximab, a well-known chimeric anti-CD20 mAb IgG1 isotype, mediated ADCC against CLL cells with a mean lysis = 34% ± 2 (n=6; Fig. 4 ). ADCC in the presence of the anti-CCR7 IgM was not tested, as this isotype is not efficacious in mediating this kind of cytotoxicity.


Figure 4
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  		Figure 4. Murine anti-CCR7 IgG2a mAb does not mediate ADCC of CLL cells, which when previously incubated with anti-CCR7 IgG mAb (2 µg/mL), the IC, or rituximab (as positive control), were exposed during 4 h to human NK cells at E:T ratios between 5:1 and 40:1. Then, dead cells were determined by 7-AAD incorporation and FCM analysis in electronically gated CLL cells. Bars show mean ± SD of six experiments.

 
Apoptosis and proliferation in response to anti-CCR7 mAb
Growing evidences suggest that a substantial portion of the beneficial responses to the therapeutic mAb is a result of direct effects on cell cycle or survival of the target cells. The effect of the anti-CCR7 mAb on the apoptosis of CLL cells was studied by 7-AAD staining of the cellular DNA and analyzing the sub-G1 peak. The rate of apoptosis of CLL cells treated for 24 h with soluble anti-CCR7 mAb was not significantly different from apoptosis of CLL cells treated with an IC (data not shown). Similarly, cross-linking of CCR7 by plate-binding of anti-CCR7 mAb up to 48 h did not result in an induction of apoptosis (data not shown).

To evaluate whether anti-CCR7 mAb induce any change in cellular proliferation, classical [3H]-thymidine uptake proliferation assays were performed with PBMC from healthy donors, as CLL cells present a high spontaneous rate of apoptosis after 48–72 h of incubation. These assays revealed a moderate increase in [3H]-thymidine incorporation in cells after incubation with anti-CCR7 IgM mAb, which was not significantly different from the one obtained with its IC. Conversely, neither the anti-CCR7 IgG mAb nor its IC induced proliferation (Fig. 5 ). As positive control, the PBMC proliferated in response to anti-CD3 mAb plus IL-2.


Figure 5
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  		Figure 5. Anti-CCR7 mAb do not induce proliferation of lymphocytes. PBMC from healthy donors were cultured for 72 h with the anti-CCR7 mAb, ICs, anti-CD3 mAb plus IL-2, or medium alone. DNA synthesis in the final 16 h of culture was determined by [3H]-thymidine incorporation. Bars represent mean ± SD of six cases performed in triplicate. cpm, Counts per minute.

 
Anti-CCR7 mAb inhibit the in vitro migration of CLL cells
CCR7 plays a main role, not only in the physiological homing of naïve lymphocytes and mature DC but also in the metastatic migration of cancer cells expressing it and particularly, CLL cells. Neutralizing the function of this chemokine receptor could be essential in CLL patients to block the dissemination of the disease to the lymphoid tissue. In this regard, we have assayed the ability of anti-CCR7 mAb to neutralize the in vitro migration of CLL cells in response to CCL19. The anti-CCR7 IgG mAb was effective in blocking the migration of CLL cells toward CCL19, whereas the anti-CCR7 IgM mAb decreased it to a lesser extent (Fig. 6 ). In these assays, migration of the cells in response to CXCL12 (ligand of the chemokine receptor CXCR4) was not affected by either of the two mAb used.


Figure 6
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  		Figure 6. Anti-CCR7 mAb block the migration of CLL cells in response to CCL19. Chemotaxis assays were performed with CLL cells as described in Materials and Methods after preincubation of the cells for 30 min with 2 µg/mL anti-CCR7 IgG (solid bars) or anti-CCR7 IgM (shaded bars) mAb or without mAb (open bars). Migration in response to CCL19 (1 µg/mL) was compared with basal migration and migration in the presence of CXCL12 (100 ng/mL), which was not affected by anti-CCR7 mAb. Bars show mean ± SD of six cases.

 
DISCUSSION

Many mAb have been evaluated for their therapeutic efficacy in the treatment of different hematological malignancies [6 ] in an attempt to develop a more specific therapy than the conventional chemotherapy. However, only few of these mAb, alone or in combination, have been demonstrated to be effective.

The humanized anti-CD52 mAb (alemtuzumab, Campath-1H) is the mAb with the greatest activity when used as single agent in CLL. The anti-CD52 mAb has demonstrated to be useful in fludarabine-resistant patients, albeit with a brief duration of response and low rates of complete remissions. In addition, the use of alemtuzumab implies a significant risk of developing opportunistic infections, as CD52 is highly expressed in every leukocyte subpopulation [39 ]. Rituximab, a chimeric anti-CD20 mAb, is not effective in CLL when used as a single agent [40 ]. However, a high rate of complete remissions has been obtained with a combination regimen including rituximab and fludarabine, with or without other chemotherapeutic agents [1 2 3 4 5 ]. Nevertheless, in spite of such promising results, CLL remains incurable to date.

Overall, these data underscore the importance of developing more specific treatments to obtain the best initial response in hematological malignancies and in particular, in CLL. An ideal surface target for immunotherapy in cancer is a molecule which is highly and specifically expressed by the malignant cells and where binding by antibodies or other molecules does not induce cellular proliferation or internalization of the molecule itself.

Previous data from our group [9 ] showed that the levels of expression of the chemokine receptors CCR7, CXCR4, and CXCR5 on B cell lymphoproliferative disorders are heterogeneous depending on the histological subtype, and significantly related to LN involvement, as these molecules mediate the entry and positioning of lymphocytes into the secondary lymphoid tissue. The expression of CCR7, CXCR4. and CXCR5 is especially high in CLL and mantle lymphoma cells, explaining the high tendency of these diseases to invade LNs. Similarly, recent studies have reported the expression of CCR7 on malignant cells from different hematological malignancies such as T cell leukemia [11 ], Hodgkin disease [12 ], and mycosis fungoides [13 ] or nonhematological solid tumors such as melanoma [14 ], breast cancer [15 ], gastric cancer [16 ], esophageal squamous cell carcinoma [17 ], nonsmall cell lung cancer [18 ], and squamous cell carcinoma of the head and neck [19 ]. It is interesting that this expression correlates, in all the cases, with a characteristic pattern of migration and metastasis into the lymphoid tissue.

These data suggest the possibility of using as therapeutical targets in immunotherapy the chemokine receptors CXCR4, CXCR5, and particularly, CCR7, not only as a result of its high density in certain malignant cells and its restricted expression in normal tissues but also because of its crucial role in the progression of cancer. In this regard, other studies have shown promising results using anti-CXCR4 mAb or antagonist peptides in some tumor models, despite the broad expression of this chemokine receptor [41 , 42 ], and also, another chemokine receptor, CCR4, has been studied [43 ] as a target in immunotherapy of T cell leukemias and lymphomas as a result of its association with skin involvement of these diseases.

Even with the successful and increasing use of antitumor mAb, the relative in vivo contribution of their different mechanisms of action is not well known. The most widely studied example, rituximab, kills target cells through the activation of complement [38 , 44 45 46 47 48 49 ] and cytotoxic cells [38 , 50 ] and has been demonstrated recently to have direct effects on the induction of growth arrest or apoptosis [51 , 52 ]. Cooperative effects cannot be excluded, such as the deposition of C3b fragments followed by opsonization and enhanced phagocytic and cytotoxic activity of the different effector cells.

Additional factors influence the therapeutic activity of mAb, including the vascularization of the tumors. Hence, blood-borne leukemic tumors can be easily accessible to complement proteins, whereas solid tumors may require the penetration of effector cells to kill them [48 , 53 ]. Finally, mAb, directed to the same target antigen, may eliminate tumor cells through different mechanisms of action [54 , 55 ].

In the particular case of CLL, ADCC does not seem to play a predominant role in eliminating tumor cells as a result of the unfavorable effector-to-tumor cell ratio, the functional defects found in the T and NK populations [56 , 57 ] of these patients and the lack of correlation between FcR for IgG (Fc{gamma}R)IIIa and Fc{gamma}RIIa polymorphisms and the therapeutic response to rituximab or alemtuzumab [58 , 59 ]. These findings together with the predominant leukemic character of CLL led us to evaluate the ability of anti-CCR7 mAb to fix complement and mediate cytotoxicity against CLL cells. Our results show an important lysis of CLL cells as a result of complement fixation with little damage of normal leukocytes even under conditions of saturating concentrations of anti-CCR7 mAb and complement. This could be a result of the lower expression of CCR7 in these cells when compared with CLL cells. In this regard, we found a close correlation (P=0.025) between the CCR7 density on the surface of CLL cells and the percentage of lysed cells under the same experimental conditions. It is important that these results suggest that the treatment of CLL with anti-CCR7 mAb, even at low concentrations, may result in an effective elimination of the tumor cells without lysing normal lymphocytes and DC expressing the molecule. It will be interesting to assess if normal leukocytes expressing CCR7 are eliminated with this treatment and in this case, to evaluate the potential undesirable effects of the treatment. We can hypothesize that the immunodeficiency secondary to the treatment with an anti-CCR7 mAb would not be very important, as CCR7-negative effector lymphocytes would remain undisturbed. The extrapolation of the immunological deficiencies that characterize the CCR7-deficient mice [60 ] to CLL patients treated with anti-CCR7 mAb is somehow difficult, as these individuals have already developed immunological memory that is absent in the knockout mice for CCR7.

The influence of clinical and prognostic factors in the sensitivity of CLL cells to CDC should be analyzed in a larger group of patients. According to our data, the only factor that affects the magnitude of CDC is the expression levels of CCR7, which are not related to the stages of the disease in apparent contradiction with Ghobrial et al. [61 ]. The discrepancy could be related to the low number of patients in advanced stages and the different technical approaches.

Unlike the findings regarding CDC, the anti-CCR7 IgG mAb studied was not an activator of ADCC, probably as a result of its murine origin and the IgG2a isotype. However, even a chimeric mAb such as rituximab does not mediate an important ADCC of CLL cells, and a significant effect is only obtained when high E:T ratios are used. Nevertheless, molecular engineering techniques or different mAb expression systems are useful to obtain and optimize the desired characteristics of a therapeutic mAb [62 , 63 ].

Blocking the function of the target antigen constitutes another mechanism of action of therapeutic mAb. In our case, blocking anti-CCR7 mAb may have the additional advantage of inhibiting the function of the probably main molecule involved in the nodal dissemination of lymphoid tumors and other nonhematological malignancies expressing it. In this regard, it is well known that neither rituximab nor alemtuzumab is effective in reducing lymphadenopathies in CLL patients.

Our results open a new therapeutic chance for the pathologies in which a blocking anti-CCR7 mAb could impair the migration to LNs and thus block the tumor dissemination in addition to killing cells by CDC or ADCC.

ACKNOWLEDGEMENTS

Grant Number 020663 from the Fondo de Investigaciones Sanitarias del Ministerio de Sanidad y Consumo, Spain, to C. M. supported this work. S. L-G. is supported by the Fundación LAIR, Spain. We thank Antonia Rodriguez, Ana Ramirez, and Mariano Vitón for excellent technical assistance and Drs. M. Gómez and A. Valenzuela for critical reading of the manuscript.

Received November 2, 2005; revised February 9, 2006; accepted February 22, 2006.

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