We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.
Rituximab, a chimeric monoclonal antibody (mAb) that binds to CD20, has greatly improved therapy for B-cell malignancies (BCM) including non-Hodgkin's lymphoma and chronic lymphoblastic leukemia.1, 2, 3, 4 Nevertheless, a considerable percentage of patients are refractory to treatment with rituximab and relapse after an initial response. Several resistant mechanisms have been proposed including escape into CD20-negative cells by limited surface antigen renewal, cell membrane drug efflux pumps, escape into the resting phase of the cell cycle, enhancement of complement inhibitory factors, alterations in intracellular signaling or cell death pathways, FcγRIIIA polymorphism and reduction of effector cells.5 Above all, downregulation of CD20 and enhancement of complement inhibitory factors plays an important role in acquired resistance to rituximab. Investigation of complement inhibitory factors demonstrated that CD55 plays an important role as a regulator of complement-dependent cytotoxicity (CDC) in malignant B-cells, and that its expression correlated with resistance to CDC,6, 7 which is one of the main mechanisms of action in the treatment of rituximab.
To overcome resistance to rituximab, several new agents have been developed, including radioimmunotherapy and mAbs against targets other than CD20.2, 8, 9 Among them, CMC-544 has been introduced as a promising agent to treat refractory/resistant BCM. CMC-544 is a conjugate of N-acetyl γ-calicheamicin dimethyl hydrazide (NAc γ-calicheamicin DMH) and a recombinant humanized antibody (IgG4) directed against the CD22 antigen.10 Calicheamicin, a very potent antitumor antibiotic agent, binds to the minor groove of DNA in a sequence-specific manner and breaks double-stranded DNA.11 Preliminary data from ongoing clinical trials reveal that CMC-544 is efficacious against recurrent/refractory B-cell lymphomas with manageable thrombocytopenia reported as the most significant toxicity.12
Concomitant use of CMC-544 and rituximab is an ideal therapeutic method because these agents have a different target molecule and mechanism of action. In fact, the additive combination efficacy of CMC-544 and rituximab was shown against xenogenic BCM in severe combined immunodeficient mice.13 However, the mechanism of the combination efficacy and the best administration schedule have not yet been elucidated. In this study, we attempted to clarify them from the viewpoint of the alteration of target molecules, that is, CD20, CD22, CD55 and CD59, which are essential for the action of these mAbs.
Materials and methods
CD22-positive cell lines used were: human lymphoma cell lines, Daudi and Raji, and their mdr-1 DNA-transduced sublines, Daudi/MDR and Raji/MDR.14 Daudi/MDR and Raji/MDR had detectable mdr-1 messenger RNA (mRNA) and P-glycoprotein. The CD22-negative cell lines used were K562 (Riken Cell Bank, Tsukuba, Japan), Jurkat (Riken Cell Bank) and NB4 (kindly provided by Dr M Lanotte, Hospital Saint-Louis, Paris, France). These cell lines were cultured in RPMI-1640 supplemented with L-glutamine (2 mM), antibiotics and 10% fetal calf serum (FCS) (Gibco BRL, Grand Island, NY, USA) (10% FCS-RPMI) at 37 °C in a humidified 5% CO2 incubator.
After informed consent, malignant cells were obtained from 12 patients with BCM. Lymphocytes were collected from the peripheral blood of eight patients with chronic lymphoblastic leukemia. Lymphoma cells were separated from the lymph nodes of four patients with large B-cell non-Hodgkin's lymphoma, and purified by density gradient with Ficoll-Paque (Pharmacia, Uppsala, Sweden).
Flow cytometry for CD20, CD22, CD55 and CD59
For the detection of CD20, CD22, CD45, CD55 and CD59, cells were stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated anti-CD20, anti-CD22, anti-CD55 or anti-CD59 mAbs in addition to Cy7-conjugated anti-CD45 mAb (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), according to the manufacturer's instructions.15 Ten thousand events were counted and mean fluorescence intensities (MFIs) were calculated using an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA). All measurements were performed in triplicate.
Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for CD20, CD22, CD55, CD59 and GAPDH mRNA in Daudi and Raji cells
The levels of CD20, CD22, CD55, CD59 and GAPDH mRNA in Daudi or Raji cells were measured by real-time RT-PCR. In brief, total cellular RNA was isolated using an RNeasy Plus Mini kit (Qiagen, Tokyo, Japan). The extracted RNA was reverse transcribed by random primers and Super Script III reverse transcriptase (Invitrogen Japan, Tokyo, Japan). Complementary DNA (cDNA) fragments were amplified by PCR using designed specific primers: forward (5′-IndexTermCTCTCTGGGGAGGCATTATGTA-3′) and reverse (5′-IndexTermGTAACAGTATTGGGTAGATGGGGAG-3′) for CD20; forward (5′-IndexTermCAGAATACATTCACGCTAAACCTG-3′) and reverse (5′-IndexTermAACACTGGGGTTACTGGAATTGTA-3′) for CD22; forward (5′-IndexTermTTCAGGCAGCTCTGTCCAGTG-3′) and reverse (5′-IndexTermGAGGCTGAAGTGGAAGGATCG-3′) for CD55; forward (5′-IndexTermCTGTGGACAATCACAATGGGAATGGGA-3′) and reverse (5′-IndexTermGGTGTTGACTTAGGGATGAAG-3′) for CD59; and forward (5′-IndexTermAAGGTCATCCCAGAGCTGAA-3′) and reverse (5′-IndexTermATGTCATCATACTTGGCAGGTT-3′) for GAPDH using Power SYBR Green PCR Master Mix kit (Takara Bio Inc., Kusatsu, Japan) with an automatic 7500 Fast Real-Time PCR System (Applied Biosystems, Tokyo, Japan).16, 17, 18 The data were expressed on a log scale as the relative expression to GAPDH. All measurements were performed in triplicate.
Monoclonal antibodies and NAC-calicheamicin DMH
Humanized IgG4 anti-CD22 mAb (G5/44) and NAc-γ-calicheamicin DMH conjugated one (CMC-544) as well as calicheamicin-conjugated humanized IgG4 anti-CD33 mAb (GO) and unconjugated NAc-γ-calicheamicin DMH were kindly provided by Wyeth Research (Collegeville, PA, USA). Rituximab was purchased from Zenyaku Co. (Tokyo, Japan).
Cell cycle distribution analysis
Cells suspended in 1 ml hypotonic fluorochrome solution containing propidium iodide (PI) (Sigma, St Louis, MO, USA) were analyzed by flow cytometry as described earlier15, 19 after incubation with CMC-544 containing 1–100 ng/ml calichemicin DMH for 72 h.
Morphological analysis by video-microscopic technique
Cells were plated in a glass-bottomed dish (MatTec Corporation, Ashland, MA, USA) at a concentration of 105 cells per ml in a medium containing CMC-544 (10 ng/ml calichemicin DMH) or an equivalent amount of G5/44. After 12 and 24 h incubations at 37 °C, cells were observed under an inverted Normarski microscope (Axiovert 35; Zeiss, Oberkochen, Germany) as described previously.19
Cells were incubated with CMC-544 or G5/44 for 12–24 h at 37 °C before staining with fluorescence-labeled mAbs (described in Flow cytometry for CD20, CD22, CD55 and CD59). Cells were placed on a non-fluorescent glass slide and observed by the C1si real spectral imaging system (Nikon Instech, Kawasaki, Japan). The fluorescence of FITC and phycoerythrin were detected simultaneously.
Dye exclusion test with propidium iodide (PI) staining
After the incubation of cells with CMC-544, G5/44 or rituximab for the indicated periods of time, cells were stained with 0.1 μg/ml PI solution and counted under the microscope.20 Viable cell counts were calculated as follows: (viable cell count)=(total cell count)−(PI-stained cell count).
Direct antiproliferative and apoptotic effect of CMC-544
Three possible mechanisms responsible for the combined effect of CMC-544 and rituximab were investigated separately: direct antiproliferative and apoptotic effects of the mAb, CDC and antibody-dependent cellular cytotoxicity (ADCC). These assays were conducted after the incubation of Daudi and Raji cells with CMC-544 either in the presence or absence of rituximab.
The antiproliferative effect of rituximab in the presence and absence of CMC-544 or G5/44 was determined by a viable cell count in triplicate. The apoptotic effect was analyzed by cell cycle distribution. In brief, 106 per ml viable cells were incubated in the presence or absence of CMC-544 containing 1–10 ng/ml calichemicin DMH for 2 h, washed three times and then incubated with or without 20 μg/ml rituximab for 72 h. Separately, all of the cells were incubated in the presence of 10 μl per 106 cells of anti-human IgG goat antibody F(ab)2 (Becton Dickinson Immunocytometry Systems) to enhance the cross-linking effect of rituximab on the cell surface.
The CDC effect was measured by a dye exclusion test in triplicate. After 106 per ml cells were incubated with or without rituximab in the presence of fresh human AB serum for 2 h at 37 °C, cells were placed on ice to stop the CDC reaction. Viable cells were counted immediately after incubation and compared with those counted before incubation.21
The enhancement of the CDC effect was studied in a similar way in the presence of CMC-544 or G5/44. Specifically, after cells were incubated with or without CMC-544 (5 ng/ml calichemicin DMH) or G5/44 at 37 °C for 2 h, they were washed three times to remove unbound antibodies. The viability of cells before incubation with CMC-544 was 99.8%. After the cells were re-incubated in CMC-544- and rituximab-free medium at 37 °C for 0–48 h, CDC was analyzed as described above.
After the first 30 min of the CDC assay, a part of the cells was placed on ice to stop the reaction, and analyzed the compliment deposition on the cells. This was determined by flow cytometry after staining with FITC-anti-human C3c rabbit antibody (Dako, Glostrup, Denmark) according to the manufacturer's instructions. MFIs were compared among the groups.
Antibody-dependent cellular cytotoxicity assays
Total mononuclear cells were obtained by Ficoll-Paque centrifugation and natural killer cells were enriched to 78–91% by an Easy Sep human positive natural killer cell kit (Invitrogen). Concurrently, 106 per ml target cells were stained with PKH67 using a MINI67 cell linker kit (Sigma).22 After the PKH67-stained target cells (PKH67+) were incubated in the presence or absence of CMC-544 containing 5 ng/ml calichemicin DMH or equivalent amount of G5/44 for two hours and washed three times, they were co-cultured with 104 per ml natural killer cells in triplicate with or without 2 μg/ml rituximab for 4 h at 37 °C. Then the cells were stained with 0.1 μg/ml PI solution and analyzed by flow cytometry. Target cells damaged by ADCC were classified as PKH67+PI+ cells, whereas viable target cells were classified as PKH67+PI− cells.
The data of real-time PCR and CDC, shown as means±s.d., were analyzed and compared using the Student's t-test. MFIs from flow cytometry were analyzed by paired t-test.
CD20, CD22, CD55 and CD59 expression on cells before and after incubation with CMC-544 as analyzed by flow cytometry
CD20 and CD22 were expressed on more than 99% of Daudi and Raji cells. CD55 was expressed on 72 and 57% of these cells, respectively. They were similarly expressed on Daudi/MDR and Raji/MDR cells (71 and 59%, respectively). However, CD20 and CD22 were not expressed on Jurkat, K562 or NB4 cells (data not shown).
To assess the effect of CMC-544 on antigen expression, cells were cultured for 2 h in a medium containing CMC-544 or G5/44, switched to an antibody-free medium and then examined at various time points thereafter to determine the levels of CD20, CD22, CD55 and CD59 on the cells. After 12–24 h, the level of CD22 on CMC-544-treated cells had decreased relative to that on G5/44-treated cells (Figure 1a) and continued to decrease, with the reduction of cell viability determined by PI staining (83 and 47% at 48 and 72 h, respectively in Daudi cells), and with apoptotic morphological changes determined by microscopic observation (38 and 64% at 48 and 72 h, respectively in Daudi cells). In contrast, the level of CD20 remained constant or increased after 24 h (Figure 1a), but decreased at 48 h (data not shown). Thereafter the level of CD55 significantly decreased after 12–24 h, and continued to decrease further. However, the level of CD59 did not change significantly. The levels of CD20, CD22, CD55 and CD59 on Daudi/MDR and Raji/MDR cells did not change after incubation with CMC-544 (data not shown). Further, the levels of these antigens did not change after incubation with G5/44 or GO (data not shown).
CD20, CD22 and CD55 expression on cells before and after incubation with CMC-544 as analyzed by laser microscopy
The levels of CD20, CD22 and CD55 on Daudi cells were also analyzed by laser microscopy. The levels of antigens before and after exposure to CMC-544 or G5/44 were compared (Figure 2a). Although the level of CD20 did not change significantly, that of CD22 and CD55 significantly decreased after 24 h when compared with the controls. These results are compatible with the data analyzed by flow cytometry. Similar results were obtained in Raji cells (data not shown).
CD20, CD22, CD55 and CD59 expression on cells before and after incubation with CMC-544 as analyzed by real-time RT-PCR
After incubating in a medium containing CMC-544 or G5/44 for 2 h, cells were cultured in an antibody-free medium and harvested at 1-, 3-, 6- and 12-h time points. The levels of CD20, CD22, CD55 and CD59 mRNA in Daudi or Raji cells were measured by real-time RT-PCR and normalized to GAPDH mRNA level (Figure 2b). The levels of CD22 and CD55 mRNA in CMC-544-treated cells began to decrease at 6-h and was significantly lower at 12-h. On the other hand, the levels of CD20 and CD59 mRNA did not change significantly. These changes observed in Daudi and Raji cells were not in Daudi/MDR and Raji/MDR cells. G5/44 had no effect on these mRNA levels.
CD20, CD22, CD55 and CD59 expression on patients' samples of BCM before and after incubation with CMC-544 as analyzed by flow cytometry
Quantitative alterations of CD20, CD22, CD55 and CD59 levels were also analyzed in samples from 12 patients with BCM. The level of CD22 decreased after incubation with CMC-544 in the samples from all patients (P<0.001). The level of CD20 was constant in the samples from four patients but increased in eight patients. Although the level of CD55 decreased significantly (P<0.001), that of CD59 did not decrease (P=0.096). Results derived from four representative samples are shown in Figure 1b. Cell sizes increased in all samples after 24 h incubation as determined by a two-dimensional analyses of forward and side scatter (Figure 3a). The enlargement was confirmed by morphological changes observed by microscopy (Figure 3b).
Direct antiproliferative and apoptotic effects of rituximab in combination with CMC-544
The antiproliferative effect was determined by a viable cell count after incubation with either rituximab, CMC-544 or both agents (Figure 4a). Viable cell counts after incubation with rituximab, CMC-544 or both agents compared with those before the incubation were 93, 45 and 41% in Daudi cells, and 96, 37 and 34% in Raji cells, respectively.
The apoptotic effect was investigated by cell cycle after incubation with either rituximab, CMC-544 or both agents (Figure 4a). The hypodiploid portion after incubation with rituximab, CMC-544 or both agents increased 4, 35 and 38% in Daudi cells, 8, 56 and 60% in Raji cells, respectively.
Thus, the antiproliferative and apoptotic effects of rituximab alone were significantly less than that of CMC-544 alone. The same results were obtained in BCM cells. The antiproliferative and apoptotic effects were not observed in incubation with G5/44 (data not shown).
Combination effect of CMC-544 on CDC caused by rituximab
The viable cell count significantly decreased after incubation with rituximab through CDC in Daudi and Raji cells. However, no effect on CDC was observed with CMC-544 or G5/44 alone (Figure 4b). Furthermore, the CDC caused by rituximab was not enhanced by simultaneous incubation with CMC-544 or G5/44.
To determine if CDC is enhanced by the sequential incubation of rituximab and CMC-544, Daudi and Raji cells were first incubated in a medium containing CMC-544 for 2 h, incubated for 12 h in an antibody-free medium, and then CDC by rituximab was analyzed. The CDC effect of rituximab was significantly increased 12 h after incubation with CMC-544 (P<0.001) (Figure 4b). G5/44 did not increase the CDC effect of rituximab. Similar results were obtained from patients' samples (Figure 4c).
After the first 30 min of the CDC assay using rituximab and AB serum, compliment deposition on the cells was analyzed by flow cytometer. The level of C3 on CMC-544-treated cells increased to G5/44-treated cells significantly (P=0.034) (Figure 4d).
Combination effect of CMC-544 on ADDC caused by rituximab
We counted viable target cells, that is, PKH67+PI− cells, and compared them before and after incubation with rituximab or CMC-544 in Daudi and Raji cells. Forty-nine percent of PKH67+PI− cells changed to PKH67+PI+ after a 4 h incubation with rituximab, whereas 9 and 2% changed after incubation with CMC-544 or G5/44, respectively. The change was not significant for simultaneous incubation with rituximab and CMC-544 (53%) or G5/44 (51%) (P=0.11 and P=0.21, respectively) nor was it increased by sequential incubation with rituximab and CMC-544. Similar results were obtained from the patients' samples (data not shown).
Rituximab has provided many encouraging clinical outcomes in the treatment of BCM.23 CMC-544, recently introduced, is also a promising agent for BCM. These mAbs target different antigens and have different antitumor mechanisms. We investigated the effects of these agents from the viewpoint of alteration of the target molecules, that is, CD20, CD22, CD55 and CD59, which are essential for their action, and attempted to clarify the rationale for the advantage in combination of rituximab and CMC-544.
CD20 is a good target for BCM because it is expressed at high levels and is not downregulated after antibody binding.2 Although malignant B-cells are heterogeneous and multi-step events occur before and after binding to CD20, the level of CD20 expression is presumed to be an important factor in treatments with rituximab,3, 5 and upregulation of CD20 has been attempted by some investigators.24 In this study, the level of CD20 was constant or increased 12–24 h following exposure to CMC-544 as analyzed by flow cytometry, and constant by real-time RT-PCR. The preservation of CD20 supports the efficacy of rituximab after treatment with CMC-544.
The levels of CD20 began to decrease over time after 24 h. This might be explained by the direct antiproliferative and apoptotic effects of CMC-544, which started 24 h after incubation with CMC-544. A similar observation was reported in our study of GO.15, 19, 20
Although the level of CD20 mRNA did not change as determined by RT-PCR, the levels of CD20 surface antigen increased as determined by flow cytometry in some samples. This discrepancy might be explained by the increase in cell volume associated with cell cycle events such as calicheamicin-induced transient G2/M arrest.15 The increase was confirmed by microscopic inspection and scatter-grams of flow cytometry.
The levels of CD22 are also important in the action mechanism of CMC-544 because CD22 directly reacts with mAb moiety of CMC-544. In our study, CD22 decreased 12 and 24 h after CMC-544 exposure as measured by RT-PCR and flow cytometry, respectively. The level of CD22 did not decrease after G5/44 or GO exposure (data not shown). Therefore, the downregulation of CD22 could be an effect of CMC-544, probably induced by a calicheamicin detached from CMC-544.
CD55 and CD59 are important regulators of CDC in BCM.6, 7 The increase of these antigens is reportedly related to their resistance to rituximab, and the decrease to their susceptibility to rituximab.21, 25 However, the underlying mechanism for the increase of these antigens with respect to the resistance to rituximab has not been well elucidated. Decreasing or inactivating CD55 is an effective idea to restore the therapeutic effect of rituximab.8 In our present analyses, the level of CD55 significantly decreased 12–24 h after CMC-544 exposure. It is consistent with the observation that CDC from rituximab increased 12 h after incubation with CMC-544. These results may also explain the rationale for the advantage in combination of rituximab and CMC-544. The increase of compliment deposition on CMC-544-treated cells also supports this advantage. Although, in this assay, some cases did not show an increase of compliment deposition, their viable cell counts had already decreased to 87–71% after the first 30 min of CDC assay. The cells that trapped more compliment deposition and were susceptible to CDC might have been damaged in the early phase of CDC assay. In this study, we could not show the combination efficacy of rituximab and CMC-544 on ADCC as CMC-544 might be quickly internalized after binding to CD22.
We showed here the rationale for the advantage of combined use of CMC-544 and rituximab in BCM. CMC-544 preserved the level of CD20 and decreased the level of CD55, both of which are closely related to resistance to rituximab.6, 7, 8 Sequential combination of these agents, that is, CMC-544 followed by rituximab, may be a relevant way forward. Such combination approaches may enable more promising therapeutic approaches for the treatment of BCM especially in relapse or refractory to conventional treatments with rituximab.
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We express our sincere gratitude to Wyeth Pharmaceuticals Inc. (USA) for their continuous support and for reviewing the article, and to Ms Yoshimi Suzuki, Ms Noriko Anma and Dr Kiyoshi Shibata (Equipment Center at Hamamatsu University School of Medicine) for technical assistance. This study was supported by Japanese grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (19590552, 17590489).
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Takeshita, A., Yamakage, N., Shinjo, K. et al. CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia 23, 1329–1336 (2009) doi:10.1038/leu.2009.77
- chronic lymphoid leukemia (CLL)
- malignant lymphoma
- monoclonal antibody
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