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Keywords:

  • chronic lymphocytic leukaemia;
  • immunotherapy;
  • CD20;
  • direct cell death;
  • B cell depletion

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

The effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the Western world and is characterized by the accumulation of long-lived B lymphocytes. Despite recent advances in its therapy, the disease still remains incurable and new treatment options need to be developed. Among targeted therapy approaches treatment with monoclonal antibodies has the advantage of specifically attacking tumour cells and thus is usually associated with mild side effects.

As a common cell surface antigen of all B cells except stem or plasma cells, CD20 has become the most effective antibody target for the treatment of B cell malignancies (Molina, 2008) and, despite variable surface expression on CLL cells, has also been considered for CLL therapy. Thus, rituximab treatment of CLL patients leads to efficient tumour cell depletion alone (Byrd et al, 2001; Huhn et al, 2001; O’Brien et al, 2001) and particularly when combined with chemotherapeutic agents (Byrd et al, 2003; Keating et al, 2005). Together with the monoclonal anti-CD52 antibody alemtuzumab, rituximab thus may be counted among the most efficient targeted treatment options for CLL reported so far. In a recent phase III trial the inclusion of rituximab was shown to substantially improve the established fludarabine/cyclophosphamide chemotherapy regimen (Hallek et al, 2010).

Treatment with anti-CD20 antibodies leads to both, direct cell death (DCD) induction by receptor engagement and cell killing by Fc-mediated mechanisms, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Glennie et al, 2007; Lim et al, 2010). According to the epitope-binding mode two types of anti-CD20 antibodies are distinguished (Cragg et al, 2003), type I, e.g. rituximab, and type II, e.g. GA101. Treatment with type I antibodies leads to CD20 accumulation in lipid rafts and to efficient CDC induction. In turn, type II antibodies potently induce DCD and lead to homotypic aggregation.

The anti-CD20 antibody GA101 (afutuzumab, RO5072759) was developed from the murine antibody B-ly1 and found to exhibit far better pre-clinical overall anti-tumour activity than rituximab in various B cell malignancies including one CLL sample (Mössner et al, 2010). During humanization, variants at the elbow hinge region with superior antigen binding were selected, which recognize a type II epitope. In addition the Fc region was glyco-engineered in order to enhance the affinity for Fcγ receptors and thus to improve ADCC (Ferrara et al, 2006).

In contrast to its in vivo efficacy in terms of B cell depletion, rituximab shows only minimal direct cytotoxic effects on CLL cells in vitro in the absence of cross-linking (Stanglmaier et al, 2004; Zent et al, 2008). Therefore it was of interest to compare, on isolated CLL cells, the action of rituximab and the novel type II CD20 antibody GA101 with greatly improved DCD induction in various lymphoma cell lines (Mössner et al, 2010). As GA101 is a promising new therapeutic reagent for CLL and entering into clinical trials for CLL therapy (Morschhauser et al, 2009), we investigated specifically for CLL the size and distribution among individual samples of GA101-induced anti-tumour effects. For this purpose we assessed whole blood samples from 37 CLL patients for their response to CD20 antibodies in an autologous assay that indicated B cell depletion due to direct as well as Fc-mediated killing mechanisms. In most samples, GA101-dependent depletion of CLL cells was sufficient for attempting a dissection of contributing mechanisms by specific interference. For this purpose we specifically targeted FcγRIIIa, also known as CD16, which is displayed on natural killer (NK) cells and macrophages, because ADCC activity in peripheral blood monocytes is dominated by NK cells (Taylor & Lindorfer, 2008). Other reasons for choosing CD16 among the Fcγ receptors displayed on various effector cell populations include the occurrence of extensively explored low and high affinity variants of FcγRIIIa (Cartron et al, 2002) and its importance for Fc glyco-engineering (Bowles et al, 2006; de Romeuf et al, 2008). As GA101-dependent B cell depletion showed high inter-individual variation we stratified the investigated CLL samples according to prognostic markers and investigated the possibility of correlations between these molecular features of CLL samples and antibody effects with the aim of identifying the patient subgroups which benefit most from GA101 treatment. Given that B cell depletion correlated with CD20 expression on CLL cells, we rationalized that upregulation of CD20 expression via engagement of TLR9 by CpG-containing oligonucleotides (CpG-ODN) might improve antibody-mediated B cell depletion and tested this hypothesis. In addition we investigated in vitro the combination treatment with anti-CD20 antibodies and chemotherapeutic agents, which currently is the predominant clinical application of rituximab for the treatment of CLL.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

Patient samples

Peripheral blood samples were obtained from patients who had been previously diagnosed with CLL according to standard criteria (Hallek et al, 2008b). The studies were performed in accordance with the local ethics committee of the University of Cologne (approval no. 01-163) and after informed written consent had been obtained from all patients included in the study. The clinical and biochemical characteristics of samples from 41 patients (76% male, median age 67 years, 39% untreated) are summarized in Table I. Characterization and stratification of patient samples according to molecular prognostic markers of CLL was performed as described previously (Veldurthy et al, 2008) and detailed in the Supporting information.

Table I.   Characteristics of the studied patient samples.
ID no.Sex/age (years)Stage (Binet)Treat-ment*Karyotype (cytogenetic aberrations)IGHVZAP70CD38% B cell depletion after treatment with 10 μg/ml GA101 (rituximab)
  1. *u (untreated)/t (treated): never treated/with prior treatment (more than 3 months ago); n.d.: not determined.

  2. †um (unmutated)/M (mutated): <2%/>2% sequence divergence with the closest germline gene.

  3. ‡p (positive)/n (negative): percentage of cells with higher fluorescence after staining with specific antibody than with isotype antibody >20%/<20%.

4m/68Budel 13qMnn60 (12) 52 (11)
5m/79Aun.d.umpp69 (49)
7m/69Aun.d.n.d.nn.d.7 (9)
8f/78Aun.d.n.d.nn34 (17)
9m/82Btn.d.n.d.pp34 (15)
10m/70Cttrisomy 12n.d.np88 (26) 83 (n.d.)
11m/76Ctn.d.umppn.d.
12m/80Aun.d.Mpn52 (16)
13m/56Audel 13q, del 17pumnn4 (4)
14m/69Ctdel 11q, del 13qn.d.nn7 (0)
15m/89Ctn.d.Mnn29 (11) 27 (13)
16f/86Ctn.d.n.d.pp46 (18)
17m/59Ctn.d.Mnn22 (4)
18m/64Atdel 17pumnn34 (18)
19m/69Btdel 11q, del 13qMnn15 (n.d.)
20f/52Ctnormaln.d.pn39 (14)
21m/68Atdel 13q, del 17pn.d.nn20 (11)
22m/68Btn.d.umpp21 (12)
23f/72Aun.d.n.d.n.d.n.d.72 (6)
24m/51Aun.d.n.d.n.d.n.d.23 (3)
25m/89Bun.d.n.d.nn16 (2)
26m/70Bunormalumpp57 (31)
27m/67Ctdel 11q, trisomy 12n.d.pn12 (7)
28m/66Cun.d.Mnn56 (7)
29m/46Aun.d.n.d.nn30 (11)
30m/73Ctdel17p, com-plex aberrationsn.d.pp12 (0)
31m/56Aunormaln.d.nn.d.4 (0)
32f/59Btn.d.n.d.nn50 (35)
33f/65Aun.d.n.d.nn31 (15)
34m/61AunormalMnn.d.27 (24)
35m/73AunormalMnn44 (2)
36m/73AuDel 13qMnp82 (32)
37m/65Btn.d.n.d.nn21 (20)
38m/65Ctn.d.Mpp45 (18)
39m/54Ctdel 11qumnp40 (20)
40f/45AunormalMnn24 (11)
41f/61Bunormaln.d.nn24 (0)
42m/51Ctdel 11qn.d.pn46 (0)
43f/60Cttrisomy 12n.d.pp24 (27)
44m/55Budel 13qn.d.nnn.d.
45m/47Aun.d.umppn.d.

Therapeutic antibodies and chemotherapeutic agents

Rituximab and GA101 were kind gifts of Roche Diagnostics (Penzberg, Germany). Alemtuzumab (Bayer Vital GmbH, Leverkusen, Germany) was obtained from the hospital pharmacy. Dephosphorylated fludarabine and chlorambucil were purchased from Sigma (Taufkirchen, Germany).

Cell isolation and culture

The cell line Mec-1 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Authentication included DNA-typing and immunophenotyping. Mec1 cells were derived from a CLL patient, but have undergone Epstein-Barr virus (EBV) transformation and differ from CLL samples by active proliferation and cell cycling (Stacchini et al, 1999).

The isolation, characterization and culture of primary CLL cells was performed as described previously (Veldurthy et al, 2008) and detailed in the Supporting information.

In order to stimulate TLR9 signalling in cells CLL, whole blood samples were treated with 1 μmol/l of the B-class CpG containing phosphorothioate oligodeoxy nucleotide DSP30 (TCG TCG CTG TCT CCG CTT CTT CTT GCC) (Decker et al, 2000). The sequence was custom synthesized as phos-phorothioate by TIB Molbiol (Berlin, Germany).

Proliferation and cell death assays

The XTT (sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay of cellular metabolism was performed according to the instructions of the supplier (Roche, Mannheim, Germany). Reduction of the yellow tetrazolium salt XTT was used to assess cellular respiration in antibody-treated cell suspensions and controls as detailed in the Supporting information.

For cell death assays 5 × 105 cells were collected and washed once before the determination of annexin V-binding and integrity of the cytoplasmic membrane. The cells were stained with fluorescein isothiocyanate (FITC)-labelled annexin V and 7-amino-actinomycin (7AAD; BD Biosciences, Heidelberg, Germany), and analysed using a FACS-Canto flow cytometer (BD Biosciences). The percentages of non-apoptotic cells relative to control were calculated from the percentages of viable (i.e. annexin V-negative, both 7AAD-positive and negative) antibody-treated and control cells.

Autologous whole blood B-cell depletion assay

B cell depletion from whole blood samples obtained from CLL patients was performed essentially as previously described for samples from healthy donors (Mössner et al, 2010). For this purpose, blood samples treated with or without therapeutic antibodies for 24 h were analysed flow cytometrically after staining with fluorescence-labelled anti-CD45, CD19 and CD3 antibodies according to established procedures (BD TriTEST, Cat. no. 3942443) as detailed in the Supporting information. After enumeration of the CD19-stained B cells and CD3 stained T cells, the B-cell depletion was calculated from the changes in B/T-cell ratios relative to untreated controls. For ADCC inhibition, 20 μg/ml of the anti-CD16 antibody 3G8 (Biolegend, San Diego, CA, USA) were added to whole blood samples 5 min before addition of anti-CD20 antibodies in order to block Fcγ receptors on effector cells.

CDC assay

Freshly isolated CLL cells were suspended at a density of 106/ml in complete medium containing 20% human serum (Quidel, San Diego, CA, USA) inactivated or not by incubation at 56°C for 30 min and incubated at 37°C for 4 h after the addition of 10 μg/ml of therapeutic antibodies. After staining with 7AAD, the percentage of cells showing membrane disintegration was determined by flow cytometry (Golay et al, 2000).

Statistical analysis

Error bars indicate standard deviations of the means. Significance thresholds were determined by two-tailed Student’s t-test. Regression analysis was performed according to analysis of variance (anova) procedures.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

Antibody effects on cell lines and isolated CLL cells

In several B cell lymphoma cell lines, GA101 showed superior DCD induction than rituximab (Mössner et al, 2010). Extending these studies to a CLL-derived cell line, we examined the direct effects of rituximab and GA101 on the membrane integrity and metabolic activity of Mec1 cells (Fig 1). For this purpose we assessed the antibody-induced disturbance of membrane asymmetry after 48 h of culture in the presence of 10 μg/ml rituximab or GA101 (Fig 1A). Changes of annexin V-binding alone were recorded because the antibody-induced increase of the annexin V- and 7AAD- double positive cell population was smaller. The relative increase of phosphatidylserine exposure by rituximab was marginal and rose to 10% after cross-linking cell surface-bound CD20 antibodies with mouse anti-human IgG (mah). GA101 induced phosphatidylserine exposure with higher efficiency than rituximab in Mec1 cells and was significantly relative to untreated controls; this was supported by similar observations in cultures using complete human or heat-inactivated bovine serum with and without cross-linking by mah (not shown). As further confirmation of antibody-induced DCD in Mec1 cells, we determined the influence of antibody treatment for 72 h on metabolic activity via XTT reduction, which was expected to indicate the biological effects on a proliferating cell line more sensitively than annexin V binding (Fig 1B). Relative to untreated cultures, the metabolic activity of Mec1 cells determined in the XTT assay was reduced by about 10% or 30% by 10 μg/ml of rituximab or GA101 respectively, with variable influence of cross-linking by mah in three repeat experiments. Only treatment with GA101 resulted in significant reduction of metabolic activity in the presence and absence of cross-linking in all three repeat experiments. Further dose-dependent analysis of XTT reduction in the absence or presence of functional complement showed significant antibody-mediated cytotoxicity as compared to untreated controls only for GA101 at doses above 0·3 or 3 μg/ml (Fig 1C). A trend for better efficiency in the presence of functional complement was observed for rituximab, but not for GA101. Taken together, GA101 showed stronger DCD induction in Mec1 cells than rituximab and less dependence on complement. In line with the features of a type II CD20 antibody, GA101 led to much stronger homotypic aggregation of Mec1 cells than rituximab as assessed by microscopic inspection (not shown).

image

Figure 1.  Antibody-dependent cytotoxicity on the prolymphocytic cell line Mec1. Mec1 cells were cultivated in the absence or presence of 10 μg/ml of the anti-CD20 antibodies rituximab or GA101 and analysed for annexin V binding after 48 h (A) or for metabolic activity after 72 h (B, C). Mec1 cells were cultivated in medium containing 5% heat-inactivated (A) or complete (B) human serum, or either as indicated by symbols (C). Antibody treatment was performed without and with crosslinking by 100 μg/ml of mouse anti-human IgG (mah) (A and B). Antibody-induced CDC was determined by annexin V binding (A) or XTT reduction (B, C). Error bars indicate standard deviations of viability among three independent experiments (A) or among triplicate samples (B and C) and comprise the uncertainty of untreated samples. Accordingly the significance versus untreated samples without mah was determined by paired (A) or unpaired (B and C) Student’s t-test. Experiments B and C were repeated twice with similar results.

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DCD induction by rituximab and GA101 was also investigated in freshly isolated CLL cells via annexin V binding (Figure S1). Incubation for 24 or 48 h in the presence of 10 μg of both CD20 antibodies led to slightly reduced viability in eight CLL samples. DCD induction by GA101 was consistently stronger than by rituximab. This difference was significant after 48 h of incubation, but also the inter-sample variation in the percentages of annexin V-negative cells in untreated samples was greater than after 24 h. The average decreases in viability achieved by rituximab or GA101 in eight primary CLL samples were around 6% or 11%, respectively (Fig 2A). According to paired Student’s t-test, antibody-mediated reduction in viability was significant only after treatment with GA101.

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Figure 2.  Comparison of antibody-dependent cytotoxic effects on CLL cells assayed in isolated tumour cells or in whole blood samples. (A) The percentages of annexin V-positive (both, 7AAD negative and positive) CLL cells were recorded after 48 h of culture in the presence or absence of 10 μg/ml of rituximab or GA101 and were expressed relative to the corresponding untreated samples. (B) B cell depletion from whole blood samples by the same antibody concentration after 24 h of treatment was determined according to the reduction of B/T cell ratios. In the box plot presentation empty diamonds indicate means and filled diamonds outliers. Treatment with rituximab and GA101 was compared using paired Student’s t-test.

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B cell depletion in whole blood samples from CLL patients

Rituximab-induced DCD, particularly in primary CLL cells, constitutes only a minor fraction of overall B cell depletion (Voso et al, 2002). Therefore we investigated the depletion of CLL cells from whole blood samples by treatment with anti-CD20 antibodies, because this assessment comprises Fc-mediated mechanisms in addition to DCD. Moreover, such an assay depends on the individual host-specific capacity of patient samples to mediate CDC or ADCC with potential for predicting clinical responses. For this purpose we enumerated B and T lymphocytes in antibody-treated whole blood samples and untreated controls and calculated the antibody-induced B cell depletion from the relative changes of B/T ratios (Ferrara et al, 2006). The mean B cell depletion after treatment with 10 μg/ml of rituximab and GA101 in whole blood samples from 37 CLL patients was 13% and 34% respectively, whereas the same antibodies decreased the viability of freshly isolated CLL cells by 6% and 11%, respectively (Fig 2). Thus, much greater effects of treatment with CD20 antibodies were observed in the whole blood assay than by analysis of annexin V binding in isolated CLL cells. According to these assays, GA101 exhibited significantly enhanced DCD on isolated CLL cells but greatly superior antibody-dependent CLL cell depletion than rituximab.

In order to investigate antibody-dependent B cell depletion in CLL samples, dose-response curves for rituximab and GA101 in B cell depletion assays were recorded (Figure S2) and the B cell depletion by 10 μg antibody per ml was determined in whole blood samples from 37 CLL patients (Table I). The B/T cell ratios determined in untreated whole blood samples ranged from 0·1 in healthy donors to about 200 in some CLL patients. The observed depletion did not correlate with lymphocyte counts or B/T cell ratios of untreated samples. As a validation of the B cell depletion assays from whole blood samples from CLL patients, the time course of B and T cell numbers was monitored in four CLL samples with and without antibody treatment (Figure S3).

In all investigated cases the dose-dependent B-cell depletion by GA101 was stronger than that by rituximab. Due to high GA101-dependent B cell depletion in some samples, the range of detected antibody response in different CLL samples was much wider for GA101 than for rituximab. Several blood samples from three patients were investigated for antibody-dependent B cell depletion at different times and showed reproducible patient-specific B cell depletion. When analysed patient by patient for 36 individual CLL samples, the GA101-induced B cell depletion correlated with the much smaller rituximab effects (F = 0·002). In contrast, the antibody-induced CLL cell depletion in seven whole blood samples did not correlate with the corresponding increases in phosphatidylserine exposure in isolated CLL cells from the same patients (Figure S4).

Correlation of antibody effects with the CD20 expression on CLL cells

In more than two-thirds of the investigated whole blood samples from CLL patients, treatment with 10 μg/ml of GA101 led to 20% or more antibody-mediated B-cell depletion, which occurred in less than one-third of cases after treatment with the same concentration of rituximab. The maximum B-cell depletion obtained at the same antibody concentration was 88% for GA101 and 49% for rituximab. While the antibody-induced B cell depletion from whole blood samples correlated with the CD20 surface expression on isolated CLL cells from the same patients (Fig 3A), there was no such correlation between antibody-induced phosphatidylserine exposure and CD20 expression (Fig 3B). This differential dependence of antibody effects on the CD20 density on CLL cells mirrors the lack of correlation of antibody-induced DCD and B cell depletion (Figure S4).

image

Figure 3.  Correlation of antibody-mediated cytotoxicity with CD20 expression and enhancement of both by TLR9 stimulation. Antibody-mediated B cell depletion from whole blood samples (A) or DCD induction in isolated CLL cells (B) were plotted against the CD20 expression on B lymphocytes of the corresponding individual CLL samples. Patient identification numbers are indicated for reference. (C) Increase of CD20 expression in whole blood samples from CLL patients after treatment with CpG-ODN DSP30 for 24 h. (D) Enhancement of antibody-mediated B cell depletion by treatment with CpG oligonucleotides of the same patient samples. The B cell depletion by 10 μg/ml of rituximab or GA101 was determined with and without treatment of the whole blood samples with CpG-ODNs and the effect of adding CpG-ODNs was evaluated by means of the paired Student’s t-test. MFI, mean fluorescence intensity.

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Dependence of Fc-mediated killing mechanisms on the antigen density on the target cells is the rationale for a combination therapy of anti-CD20 antibodies and CpG-ODN, which have been shown to enhance CD20 expression (Jahrsdorfer et al, 2001). Therefore we assessed the influence of treatment with the CpG-ODN DSP30 for 24 h on CD20 expression and antibody-induced B cell depletion in whole blood samples. Flow-cytometric analysis of whole blood samples after staining with CD20 antibodies revealed a significant increase of CD20 expression during 24 h of treatment with CpG-ODN (Fig 3C). The same treatment enhanced antibody-induced B cell depletion, which was significant in the case of GA101 and to a lesser degree for rituximab (Fig 3D). In isolated CLL cells from the same patients, incubation with the CpG-ODN DSP30 for 24 h slightly increased the percentage of viable, annexin V-negative cells (not shown), in line with previous reports (Decker et al, 2003). Together these findings demonstrate increased expression of the target antigen due to addition of CpG-ODN, which in turn leads to enhanced Fc-mediated antibody effects. These results also confirm that the patented enhancement of antibody effects by immuno-stimulating nucleic acids (Weiner & Hartmann, 2003) also applies for the novel CD20 antibody GA101.

Dissection of antibody-mediated killing mechanisms

CD16 is the major Fcγ receptor expressed on NK cells and interacts with the Fc portion of therapeutic antibodies mediating ADCC, which was improved in GA101 when compared to rituximab due to glyco-engineering (Mössner et al, 2010). Therefore we blocked the interaction of antibody-coated B lymphocytes with CD16 expressing effector cells in whole blood samples by addition of anti-CD16 antibodies in order to assess the contribution of ADCC to overall B cell depletion. In four freshly drawn whole blood samples from healthy donors, the mean GA101-induced B cell depletion of 45% was reduced by about one-third by incubation with anti-CD16 antibodies (Fig 4A). The rituximab-induced B cell depletion in these samples was 10% on average and declined upon pre-incubation with anti-CD16 antibody only in two of four investigated samples. In whole blood samples from five CLL patients, the average B cell depletion by rituximab or GA101 was 14% or 32% respectively, and not significantly impaired by addition of anti-CD16 antibodies, with some tendency for a decline in the case of GA101-induced B cell depletion (Fig 4B). Thus, the reduction of GA101-induced B cell depletion in healthy donors may be the result of blocking FcγRIIIa on NK cells and macrophages by anti-CD16 antibodies. The failure to detect a comparable contribution of ADCC to overall B cell depletion in blood samples from CLL patients can be explained by a much lower ratio of target to effector cells in leukaemic versus normal whole blood samples. The increased number of target cells in whole blood samples from CLL patients as compared to healthy donors corresponded to average B/T cell ratios of 52 or 0·2, respectively. In line with this concept, the blood sample from Patient 27, in which blocking of GA101-dependent B cell depletion by anti-CD16 antibody was most efficient, also showed by far the lowest B/T cell ratio of 8·3.

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Figure 4.  Contributions of ADCC and CDC to antibody-dependent B cell depletion. B cell depletion from whole blood samples obtained from healthy donors (A) or CLL patients (B) was determined after 24 h of incubation with 10 μg/ml of CD20 antibodies, with or without addition of 20 μg/ml of anti-CD16 antibody 3G8 blocking Fc-gamma receptors on NK cells and macrophages. The CDC mediated by 10 μg/ml of rituximab, GA101 or alemtuzumab was assessed flow cytometrically by 7AAD staining after incubating isolated CLL cells with therapeutic antibodies in 10% complete or heat-inactivated human serum (C).

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In order to assess the contribution of CDC to the overall cytotoxic effects elicited by therapeutic antibodies, we observed membrane integrity of isolated CLL cells 4 h after treatment with 10 μg/ml of rituximab, GA101 or alemtuzumab in the presence or absence of functional complement (Fig 4C). The percentage of 7AAD-stained cells in the presence of complement rose to about 30% after treatment with alemtuzumab, but was not substantially increased with rituximab or GA101. Selective inhibition of CDC in the B cell depletion assay with whole blood samples by addition of cobra venom factor (CVF) was complicated by unspecific effects of CVF and low rituximab-mediated overall B cell depletion (not shown).

Correlation of antibody effects with molecular and clinical features of CLL samples

As broad patient-specific differences were observed in the size of antibody-induced effects, we explored whether the response mediated by anti-CD20 antibodies was linked to molecular features of the tumour cells (Table I). Stratification according to CD38 expression showed significantly higher rituximab- or GA101-induced B cell depletion in the samples belonging to the subgroup with worse prognosis (Figure S5A). A trend for higher efficiency of CD20 antibodies in samples with high ZAP70 expression was identified, which was much less pronounced than for CD38 expression and did not reach significance (Figure S5B). Weak and strong responders to GA101 appeared equally distributed among 10 or 6 samples with or without IGHV hypermutation (Figure S5C). Stratification according to the presence or absence of deletions of chromosome 11q or 17p (del 11q or del 17p), on which the ATM or TP53 genes are located, showed a significant tendency for stronger GA101 effects in the group with apparently unaffected DNA damage response (Figure S5D). The validity of the last two correlations is, however, limited by absent IGHV mutation and fluorescence in situ hybridization data for high proportions of patient samples (Table I). The samples from patients with previous treatment were not significantly less responsive to in vitro treatment with CD20 antibodies than those from untreated patients (Figure S5E).

Combined action on CLL cells of rituximab or GA101 with chemotherapeutic agents

Given that rituximab is therapeutically beneficial especially in combination with chemotherapeutic agents, we assessed rituximab and GA101 in combination with fludarabine or chlorambucil. Although much larger antibody effects were observed in the B cell depletion assay from whole blood than DCD induction in isolated CLL cells, the latter assay format was used for assessing combinations of CD20 antibodies with chemotherapeutic agents, as it is readily compatible with both combination partners (Fig 5). In seven CLL samples rituximab or GA101 led to a mean increase in annexin V binding of 5% or 10%. The average decreases in viability induced by fludarabine or chlorambucil alone were 23% or 16%, respectively. Two samples, Samples 4 and 7, were much more sensitive to the chemotherapeutic agents than the remaining five samples, which resulted in the highest observed overall decreases in viability due to combined chemo-immunotherapy among the investigated samples. Except for the samples with the highest response to chemotherapeutic agents, all samples in Fig 5 were from patients who had been previously treated with chemotherapeutic agents. Therefore, the poor response to chemotherapeutic agents of Samples 17, 21, 22 and 27 may be explained by resistance acquired during treatment, in the case of Sample 27 additionally by del 11q. Enhancement of fludarabine and chlorambucil-induced cytotoxicity by CD20 antibodies was strongest in Sample 22, and less than additive in the Samples 7 and 17. As observed for the single agents, GA101 effects tended to be stronger than those of rituximab and resulted in a significant enhancement of chlorambucil-induced annexin V binding.

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Figure 5.  Combination of anti-CD20 antibodies and chemotherapeutic agents. The DCD induction in isolated CLL cells by the anti-CD20 antibodies rituximab and GA101 and the chemotherapeutic agents fludarabine and chlorambucil separately and in combination was determined after 48 h of treatment by flow cytometric assessment of annexin V binding. Fludarabine and chlorambucil were used at 5 μmol/l concentration, anti-CD20 antibodies at 10 μg/ml. Asterisks indicate significant enhancement of cytotoxic effects by antibodies relative to the controls without treatment or treated with the chemotherapeutic agents alone.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

The present in vitro assessment of anti-CD20 antibodies found superior cytotoxicity against CLL cells by the novel type II glyco-engineered CD20 antibody GA101 than by rituximab, the latter representing a crucial backbone in the current therapy for CLL. For this comparison we either performed cytotoxicity assays on isolated CLL cells in uniform assay matrices or observed antibody-dependent B cell depletion from whole blood samples, which supply individual host-specific immune functions.

In agreement with previous reports (Bellosillo et al, 2001; Stanglmaier et al, 2004; Zent et al, 2008) DCD induction in freshly isolated CLL cells by rituximab was variable, considerably less than in lymphoma cell lines and not reaching a statistically valid difference to untreated controls. With GA101, however, the DCD induction for both, Mec1 cells and CLL patient samples, was significantly higher than with rituximab, i.e. for primary CLL cells DCD induction was, on average, about twice as high. GA101-induced phosphatidylserine exposure on un-stimulated, resting CLL cells was less than on Mec1 cells and B cell lymphoma cell lines, but still significant. Better DCD induction in primary CLL cells and the cell line Mec1 with the type II antibody GA101 compared to the type I antibody rituximab is in agreement with observations in various lymphoma cell lines and murine xenograft models treated with the prototype type II mouse antibody B1 (tositumumab) (Cardarelli et al, 2002; Chan et al, 2003; Beers et al, 2008). Due to a lack of major antibody-mediated induction of membrane disintegration, our evaluation of cell death induction was based on phosphatidylserine exposure alone, similar to the preceding characterization of GA101 effects on B cell lymphoma cell lines (Mössner et al, 2010). Despite the observation of homotypic aggregation by GA101 in CLL cells we did not find the associated extent of membrane disintegration typical of lysosome-mediated membrane damage induced by type II CD20 antibodies in lymphoma cell lines (Ivanov et al, 2009).

Given the significantly improved but still moderate DCD induction by GA101 in primary CLL cells, we used an assay encompassing effector cell-mediated mechanisms that constitute the main contribution to rituximab-induced B cell depletion in vitro (Voso et al, 2002). In the B cell depletion assay from whole blood samples from CLL patients and healthy donors the B cell depletion by GA101 greatly surpassed that induced by rituximab. In contrast, the ex vivo depletion of B lymphocytes and externally added Raji cells from whole blood samples from healthy donors by the second generation type I anti-CD20 antibody veltuzumab (hA20) was in the same order as that of rituximab (Goldenberg et al, 2009).

GA101-induced B cell depletion was highly variable, with a range of 3–88% B cell depletion by 10 μg/ml of GA101 over 24 h. This can be partly explained by the dependence of Fc-dependent killing mechanisms on antigen density, because GA101 and rituximab-induced B cell depletion, but not DCD induction, correlated with CD20 expression on the target cells. This is in agreement with the previously observed correlation of rituximab-induced CDC with CD20 expression on CLL cells (Bellosillo et al, 2001; Golay et al, 2001). The less efficient B cell depletion by type I as compared to type II antibodies has recently been attributed to CD20 internalization, leading to reduced macrophage recruitment and to degradation of cell-bound antibodies (Beers et al, 2010).

Dissection of the contributions of DCD, CDC and ADCC to the observed B cell depletion from whole blood samples is complicated by the divergent read-out used for the assessment of B cell depletion and DCD, but mainly by numerous factors influencing host-dependent complement and immune effector cell function, most prominently inhibition of Fc-mediated antibody effects by endogenous human IgG (Lefebvre et al, 2006; Preithner et al, 2006). A substantial contribution of ADCC to overall GA101-dependent B cell depletion was shown in whole blood from healthy donors by blocking the interaction of FcγIIIa on NK cells and macrophages and the Fc exposed on antibody-coated target cells by incubation with anti-CD16 antibody. In blood samples from CLL patients the same treatment did not substantially decrease GA101-dependent B cell depletion, owing to dramatically increased amounts of target cells resulting in unfavourable ratios of effector to target cells. This effect may be enhanced by impaired NK cell levels and additional deficiencies in immune effector cell function. In the present assay matrix, the CD16-mediated ADCC of GA101 against CLL cells appeared to be much less prominent than reported for lymphoma cells (Mössner et al, 2010). A similar relationship is suggested by observations about the Phe158Val polymorphism of FcγIIIa (Cartron et al, 2002). Rituximab-induced ADCC is enhanced in about 15% of Europeans who are homozygous for the valine form expressing the high affinity variant of FcγIIIa. While the Phe158Val polymorphism of FcγIIIa predicts response to rituximab treatment for non-Hodgkin lymphoma (Cartron et al, 2002), this is not the case for CLL (Farag et al, 2004). For healthy donors our data confirm superior ADCC induction by GA101 than by rituximab (Mössner et al, 2010), which was also reported for the type II antibody mAB 1.5.3 (Bornstein et al, 2010). Quantitative comparison of ADCC elicited by different antibodies requires application of a uniform assay system, e.g. co-culture with the NK cell line NK-92 (Weitzman et al, 2009). On the other hand the B cell depletion assay from whole blood described here has the advantage of reflecting both, the efficiency of antibody-induced B cell depletion and the potential to supply host-dependent immune functions and thus should be able to predict at the individual level the clinical efficiency of therapeutics assayed in vitro.

In a similar approach as for ADCC we tried to determine the contribution of CDC to antibody-mediated B-cell depletion from whole blood samples, namely by inhibiting complement function by incubation with CVF. Possibly due to the rare occurrence of sufficiently high rituximab-induced overall B cell depletion we failed to detect its inhibition upon addition of CVF. This is in agreement with the low rituximab-mediated CDC observed in vitro in CLL cells (Bellosillo et al, 2001; Golay et al, 2001), which is considerably lower than in most B cell lymphomas (Mishima et al, 2009), and negligible in comparison with alemtuzumab-induced CDC in CLL cells (Zent et al, 2008). We confirmed the greatly differing CDC exerted by alemtuzumab and CD20-antibodies on isolated CLL cells for GA101. In Raji cells the presence of complement enhanced the cytotoxicity mediated by rituximab, but not by the anti-CD20 antibody B1 (Stanglmaier et al, 2004). Similarly, in contrast to GA101, a trend for better efficiency in the presence of functional complement was observed for rituximab in the cell line Mec1. Interestingly, the type II antibody mAB 1.5.3 has CDC comparable to rituximab for B cell lymphoma cells other than CLL (Bornstein et al, 2010).

The variability of GA101-induced B cell depletion among CLL samples poses the challenge to define molecular features that predict which patients will benefit most from this new therapeutic agent. In this context, stratification of CLL samples according to cytogenetic aberrations revealed that CD20 expression is below average for del 11q and above for trisomy 12 (Tam et al, 2008), with potential consequences for GA101 efficacy due to its relationship with CD20 expression. Indeed three of five samples in our collection with known del 11q are ranked within the quartile with lowest GA101-induced B cell depletion, while one of the three cases with known trisomy 12 exhibited the highest GA101-dependent depletion of CLL cells encountered in this study. In addition we stratified the investigated CLL samples according to CD38 and ZAP-70 expression status, both of which define prognostic subgroups with aggressive or indolent course (Rassenti et al, 2008). For both markers and, as far as can be concluded from the limited number of characterized samples, also for IGHV hypermutation, we observed a trend for stronger GA101-induced B cell depletion in the subgroups with worse prognosis. The reverse tendency, namely lower response to GA101 in the subgroup with worse prognosis, was found for cases with del 11q or del 17p as compared to samples with a different known karyotype. This also appears to apply separately to samples with del 17q, which predicts p53-deficiency, since the GA101 responses of three among the four samples with del 17p examined ranked in the lowest quartile of the investigated samples. While resistance to chemotherapeutic agents is frequently linked to previous treatment, as was the case for our samples subjected to in vitro treatment with combinations of chemotherapeutic agents and CD20 antibodies, the treatment status of the donors was not correlated with the response of whole blood samples to CD20 antibodies. This predicts that previously treated and untreated patients alike might benefit from treatment with GA101.

Since Fc-mediated killing mechanisms depend on the antigen density on the target cells, both the variability and the modulation of CD20 expression on CLL cells influenced the efficacy of rituximab and GA101. Accordingly combination therapies have been suggested that include anti-CD20 antibodies and treatments that lead to upregulation of CD20 expression, e.g. addition of the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-4. Our results extend to GA101 the previously observed CpG-ODN-induced enhancement of CD20 expression and of antibody-mediated cell killing, which can be clinically beneficial, especially in conjunction with concomitant long-term apoptosis induction (Jahrsdorfer et al, 2001). For rituximab efficacy the reverse effect due to CD20 downregulation by lenalidomide was observed (Lapalombella et al, 2008).

The predominant clinical use of anti-CD20 antibodies for treatment of CLL is in combination with chemotherapeutic agents. Therefore we assessed combinations of rituximab or GA101 with fludarabine or chlorambucil. In agreement with a previous report (Chow et al, 2002), we found enhanced action of chemotherapeutic agents on CLL cells ex vivo when combined with rituximab. The superior DCD of GA101 as compared to rituximab also translated into greater enhancements of the cytotoxicity due to treatment with fludarabine or chlorambucil. Interestingly the mutual enhancement was significant for the combination of GA101 and chlorambucil, which is under clinical investigation in the CLL11 trial of the German CLL Study Group (http://clinicaltrials.gov/show/NCT01010061).

In conclusion, the present comparison of rituximab and the novel type II anti-CD20 antibody GA101 shows better DCD-induction by GA101 in isolated CLL cells and noticeably improved antibody-dependent B cell depletion from whole blood samples. Although the improved DCD induction and ADCC reported for GA101 in B cell malignancies in general (Mössner et al, 2010) were not fully attained in CLL cells ex vivo, the size and frequency of clearly enhanced overall CLL cell depletion make GA101 a promising candidate for potential treatment strategies as a single agent or in combinations. As to the observed variability of GA101-induced effects among CLL samples, it will be interesting to compare, on an individual basis, the B cell depletion in vitro with the treatment response in larger clinical trials, with the goals of predicting treatment outcome and defining the patient subgroup that might benefit most from treatment with GA101.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

We are grateful to Reinhild Brinker and Julia Claassen for strong support in the purification and characterization of CLL cells. This study was supported in part by research funding from Roche to G.K. and M.H. This work was supported by a Research Alliance grant from the CLL Global Research Foundation to M.H. and by the Wilhelm Doerenkamp Foundation, Cologne.

Authorship and disclosures

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

G.K., M.H., C.K. P.U. and M.P conceived and designed the present work. M.P., P.I., N.F., G.K. and B.M. performed the experiments. L.F. and C.M.W. contributed patient samples and their characterization. U.P. and C.K. contributed anti-CD20 antibodies. G.K., M.P., C.K., M.H. analysed the data; G.K. wrote the paper. Potential conflicts of interest: C.K and P.U.: Employment, Equity Ownership and Patents and Royalties; M.H.: Honoraria and Research Funding; G.K.: Research Funding. The other authors (M.P., P.I., N.F., B.M., L.F. and C.M.W.) declare no competing financial interest.

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  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authorship and disclosures
  8. References
  9. Supporting Information

Fig S1. Antibody-induced DCD in primary CLL cells.

Fig S2. Dose-dependency of antibody-dependent B cell depletion from whole blood samples obtained from CLL patients.

Fig S3. Time course of antibody-mediated B cell depletion in whole blood samples from CLL patients.

Fig S4. Lack of correlation between antibody-induced B cell depletion ex vivo and posphatidylserine exposure.

Fig S5. Correlation of antibody-dependent B cell depletion with prognostic markers.

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