Novel anti-CD20 antibody TGLA with enhanced antibody-dependent cell-mediated cytotoxicity mediates potent anti-lymphoma activity

Cancer Letters 294 (2010) 66–73

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Novel anti-CD20 antibody TGLA with enhanced antibody-dependent cell-mediated cytotoxicity mediates potent anti-lymphoma activity Ming Lv a,1, Zhou Lin a,1, Chunxia Qiao a, Shusheng Gen b, Xiaoling Lang b, Yan Li a, Jiannan Feng a, Beifen Shen a,* a b

Institute of Basic Medical Sciences, Beijing, 100850, China Beijing Tian Guang Shi Biotech Co. Ltd., Beijing, 100176, China

a r t i c l e

i n f o

Article history: Received 17 December 2009 Received in revised form 20 January 2010 Accepted 20 January 2010

Keywords: Anti-CD20 monoclonal antibody TGLA Rituximab ADCC Immunotherapy

a b s t r a c t Rituximab is the first anti-cancer antibody approved by the FDA for the treatment of B-cell lymphoma. However, its efficacy remains variable and often modest. Some patients are initially unresponsive to rituximab or later develop resistance to it, and require alternative therapies. Rituximab activity has been thought to involve antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis. Present studies suggest that the patients unresponsive to rituximab may be helped with other CD20 antibodies with enhanced activities. In this study, we characterized a novel antiCD20 chimeric antibody, TGLA, which binds to various B-cell lines specially and shares an epitope with rituximab. TGLA shows equal activities with rituximab, such as CDC, cell growth arrest and so on. Interestingly, TGLA also shows significant ADCC activity. Immunotherapeutic studies further show that TGLA is far more effective in delaying tumor growth than rituximab. These findings suggest that the ADCC-enhanced anti-CD20 antibody TGLA might be an alternative therapeutic agent for B-cell lymphoma. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The CD20 antigen is present on > 90% of B-cell lymphomas and is an effective target for immunotherapeutic removal of malignant B cells [1–3]. Rituximab (C2B8) is the first anti-cancer antibody approved by the FDA for the treatment of B-cell lymphoma [4–6]. Despite the effectiveness of rituximab, only 48% of patients respond to the treatment; complete responses are <10%. In addition, a sig-

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; C2B8, rituximab; CDC, complement-dependent cytotoxicity; CHO, chinese hamster ovary; FCM, flow cytometry; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; TGLAFITC, FITC-conjugated TGLA antibodies. * Corresponding author. Tel.: +86 10 66931325; fax: +86 10 68159436. E-mail address: [email protected] (B. Shen). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.01.023

nificant number of patients have progressive disease during antibody therapy [7,8]. Therefore, alternative therapies for these patients are strongly desired. The success of more efficient mAb therapies will depend on knowledge of the precise mechanisms of action of the mAbs. We speculated that anti-CD20 mAbs with enhanced effector functions and different biological activities might potentiate the clinical response. Previous studies have suggested that several mechanisms are involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). It is also possible that the binding of rituximab to the CD20 antigen on the cell surface may directly induce apoptosis [9,10]. Although the relative contributions of these different mechanisms of action are a matter of debate, the activities of most anti-CD20 antibodies are thought to be predominantly through CDC and ADCC [11,12]. Several reports have shown that CDC enhance-

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ment, both alone and in combination with ADCC enhancement, increases the anti-lymphoma activity of anti-CD20 antibodies irrespective of individual differences in effector functions, and renders current anti-CD20 therapy capable of overcoming the potential resistance mechanisms [13– 16]. In the study, we report on a novel anti-CD20 mouse/human chimeric antibody, TGLA (Chinese patent published: CN1931877). In vitro and in vivo anti-tumor activities of TGLA were examined and compared with rituximab. TGLA bound to various B-cell lines especially and shared epitopes with rituximab. TGLA showed similar activities to rituximab, such as CDC, cell growth arrest and so on. Interestingly, TGLA showed significant ADCC activity. Results from immunotherapeutic studies further indicated that TGLA was far more effective in delaying tumor growth than rituximab. These findings suggest that the ADCC-enhanced anti-CD20 antibody TGLA might be an alternative therapeutic agent for B-cell lymphoma.

scribed previously [17]. The expression vector was transfected into CHO cells using Lipofectamine 2000 reagent (Invitrogen), after which stable transfectants were isolated by limiting dilution in the presence of 500 lg/mL G418. The cell clones producing the highest amount of TGLA were selected and grown in serum-free medium. Finally, TGLA were purified by affinity chromatography on Protein A-Sepharose (Amersham Biosciences) from the serum-free culture supernatants.

2.3. SDS–PAGE and western blot The purified proteins were analyzed on 8% SDS–PAGE under nonreducing conditions and on 12% SDS–PAGE under reducing conditions, followed by Coomassie Brilliant Blue staining. For western blot analysis, proteins were separated by SDS–PAGE under nonreducing conditions and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). After protein transfer, the PVDF membranes were treated with the blocking buffer followed by incubation with horseradish peroxidase-conjugated goat anti-human IgG (H + L; Pierce). Finally, the bands were visualized using Enhanced Chemiluminescence (Sigma).

2. Materials and methods 2.1. Cell lines, antibodies and animals Chinese hamster ovary (CHO), Raji, Daudi and Jurkat cells were obtained from the American Type Culture Collection. Rituximab was purchased from Roche Ltd. TGLA was labeled with fluorescein isothiocyanate (FITC) to produce FITC-conjugated antibodies (TGLA-FITC). Nude mice and BALB/c mice were obtained from the Institute of Zoology, the Chinese Academy of Sciences. All animal studies were performed after approval of the local animal care and use committee.

2.4. Immunoprecipitation Daudi cells were washed in PBS; lysis buffer was then added (final concentration: 0.5% Triton X-100, 50 mM Tris [pH7.6], 100 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mg/ ml each leupeptin and aprotinin, 25 mM NPGB, and 1 mM PMSF). The different anti-CD20 antibodies and the protein A-agarose beads were added to the cell lysates and incubated for 1 h at 4 °C. The samples were resolved on a 12% SDS–polyacrylamide gel and then identified by horseradish peroxidase-conjugated goat anti-human CD20 (Santa Cruz Biotechnology) using western blot.

2.2. Expression and purification of TGLA antibody The TGLA expression vector was constructed, and the antibody was expressed using identical procedures de-

A

B

Reduced 1

2

Non-reduced

3

1

55 kDa

66 kDa 45 kDa

150 kDa

26 kDa

35 kDa

120 kDa

14 kDa

100 kD kDa

C

1

2

2

3

4 155 kDa

3 155 kD kDa

Fig. 1. Characterization of TGLA. A, SDS–PAGE analysis of purified anti-CD20 mAbs under reducing conditions. Lane 1, human IgG; lane 2, TGLA; lane 3, molecular weight protein markers. B, SDS–PAGE analysis of purified anti-CD20 mAbs under nonreducing conditions. Lane 1, molecular weight protein markers; lane 2, human IgG; lane 3, TGLA; lane 4, rituximab. C, western blot analysis of purified anti-CD20 mAbs separated by SDS–PAGE. Lane 1, human IgG; lane 2, rituximab; lane 3, TGLA.

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2.5. Flow cytometry analysis Flow cytometry analysis was performed to determine the binding of the anti-CD20 antibodies to target cells using a FACScan flow cytometer (Becton Dickinson). Briefly, cells at 1  106 cells/mL were incubated with FITC-labeled recombinant antibodies for 30 min at 4 °C, or incubated with the target antibodies followed by incubation with FITC-conjugated goat anti-human IgG (Becton Dickinson). The cells were then washed and analyzed by flow cytometry (FCM). 2.6. Competitive binding assay Daudi cells at 1  106 cells/mL were incubated with a subsaturating concentration (5 lg/mL) FITC-conjugated TGLA (TGLA-FITC) and increasing concentrations of competing antibody rituximab for 30 min at 4 °C. The cells were then washed and analyzed by FCM. 2.7. Cytotoxicity assays CDC and ADCC activities of the antibodies were measured by DELFIA EuTDA Cytotoxicity Reagents (Perkin Elmer) according to the manufacturer’s instructions. Briefly, the cells were incubated with fluorescence-enhancing li-

gand, then incubated with the anti-CD20 antibodies for 1 h in culture medium in a 5% CO2 incubator at 37 °C, follow by the addition of either rabbit complement (10% vol/vol, for CDC assay) or human peripheral blood mononuclear cells (PBMC) as effector cells (effector to target, 50:1 for ADCC assay). After an additional incubation for 4 h at 37 °C, the fluorescence of the culture supernatant was measured in the time-resolved fluorometer. Maximum release was determined by lysis in 0.2% Triton X-100. Percentage of specific lysis was calculated according to the following formula: % lysis = [experimental release – spontaneous release] / [maximum release – spontaneous release]  100.

2.8. Cell growth inhibition assay The cells at 2  105 cells/mL were incubated with different concentrations of anti-CD20 antibodies in complete medium at 37 °C, 5% CO2. On the 3rd day, cell growth inhibition was evaluated by the luminescence ATP detection assay system (Perkin Elmer) according to the manufacturer’s instructions. The percentage of cell growth inhibition was calculated according to the following formula: Inhibition (%) = [(A-mAb-untreated cells – A-mAb-treated cells]/ (A-mAb-untreated cells – A-culture medium)  100.

Fig. 2. Characterization of TGLA. A, antigen binding activity of TGLA. Raji, Daudi, Jurkat or PBMC cells were incubated with TGLA-FITC for 30 min at 4 °C and then analyzed by FACS. B, TGLA binding to CD20 antigen especially. The samples immunoprecipitated from the Daudi cell lysates by different anti-CD20 antibodies were resolved on a 12% SDS–PAGE and then identified by horseradish peroxidase-conjugated goat anti-human CD20 using western blot. Human IgG is a negative control antibody. C, TGLA effectively competed with rituximab to bind to Daudi cells. Daudi cells were incubated with a subsaturating concentration (5 lg/mL) TGLA-FITC and increasing concentrations of competing antibody rituximab for 30 min at 4 °C. The cells were then washed and analyzed by FCM. Points, mean of three independent experiments.

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Fig. 3. The mechanisms of TGLA activity. A, Inhibition cell growth of anti-CD20 mAbs on Daudi and Raji cells. The cells were incubated with different concentrations of TGLA or rituximab at 37 °C, 5% CO2 for 3 days; cell growth inhibition was measured by the luminescence ATP detection assay system. Jurkat cells were used as controls. Points, mean (n = 3); bars, SD. B and C, CDC and ADCC induced by anti-CD20 mAbs. Daudi and Raji cells were incubated with increasing concentrations of TGLA or rituximab in the presence of rabbit complement or PBMCs at 37 °C for 4 h. CDC and ADCC activity of these antibodies was measured by DELFIA EuTDA Cytotoxicity Reagents. Jurkat cells were used as negative controls. Points, mean (n = 3); bars, SD; * P < 0.05.

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Fig. 3 (continued)

2.9. Stability assay

3. Results

The serum stability of TGLA was analyzed by measuring its activity after incubation in human serum at 37 °C. TGLA was diluted in human serum to a concentration of 1 mg/mL and incubated at 37 °C for varying times. At the given time points, the samples were frozen and kept at 70 °C. The binding activities to the Daudi cells of all the samples were determined by FACS.

3.1. Characterization of TGLA

2.10. Immunotherapy Groups of 5-wk-old female BALB/c nude mice were inoculated subcutaneously with 3.5  106 Daudi cells on day 0, followed 10 d later by intravenous. injection of different anti-CD20 antibodies. The mice were observed daily and euthanized at the onset of hind leg paralysis. Tumor volumes were calculated according to the following equation: Tumor volume (mm3) = 1/2  (length)  (width) 2.

The purity and the molecular weight of the purified anti-CD20 mAbs were determined by SDS–PAGE (Fig. 1). Under reducing conditions, TGLA yielded two protein bands with molecular masses of 55 kDa (heavy chain) and 26 kDa (light chain) (Fig. 1A). Under nonreducing conditions, TGLA showed a single band of 155 kDa (Fig. 1B). The identity of the purified TGLA was further confirmed by western blot using polyclonal antibodies against human IgG (Fig. 1C). These results corresponded approximately to the calculated molecular mass. The binding activity of anti-CD20 mAbs was assessed in antigen-binding assays. Flow cytometry analysis was performed using indirect immunofluorescent staining to show that TGLA binds to a panel of cultured Bcell lymphomas. As shown in Fig. 2A, TGLA bound to two human Burkitt lymphoma cell lines, Raji and Daudi, with high intensity. Discriminatingly, TGLA did not bind to the human T-lymphoma cell line Jurkat. TGLA bound to approximately 9% of PBMCs, which corresponded to data regarding the prevalence of the standard anti-CD20 antibody (data not shown).These results indicate that TGLA binds to the CD20 antigen particularly. To further confirm this conclusion, Daudi cell lysate was immunoprecipitated with TGLA or other antibody. As with rituximab, TGLA was able to precipitate CD20 in Daudi cells (Fig. 2B). In competitive binding assays, various rituximab concentrations were compared to TGLA-FITC for affinity for Daudi cells. The result shown that rituximab effectively competed with TGLA for binding to Daudi cells (Fig. 2C). Summarizing these results, TGLA possessed specificity similar to rituximab and shared the same epitope as rituximab.

2.11. Statistical analysis 3.2. Effects of anti-CD20 mAbs on proliferation of B-cell lymphomas cell lines

Statistical analysis was performed using Student’s unpaired t test to identify significant differences unless otherwise indicated. Differences were considered significant at a P value of <0.05.

Growth inhibition by the anti-CD20 mAbs was evaluated by in vitro proliferation assays in the B-cell lymphoma cell lines. Cells were cultured with mAbs; proliferation was assessed by the luminescence ATP detection assay. In both of the B-cell lines studied, specific inhibition was seen with

M. Lv et al. / Cancer Letters 294 (2010) 66–73 Table 1 The IC50 of anti-CD20 Mabs killing CD20 positive cells through ADCC activity. IC50(lg/mL)

Raji

Daudi

TGAL Rituximab

0.84 ± 0.12 6.43 ± 0.25

0.69 ± 0.03 4.72 ± 0.17

the anti-CD20 mAbs, but the level of inhibition varied between TGLA and rituximab. As shown in Fig. 3A, anti-CD20 mAbs yielded similar proliferation inhibition in the B lymphoma cell lines in small concentrations. At the mAb concentration of 50 lg/mL, TGLA was more inhibitory than rituximab. For example, Daudi cells treated with TGLA were approximately 30% inhibited, compared with approximately 22% using rituximab. Similar results were shown using Raji cells. As the negative control, specific inhibition was not seen with anti-CD20 mAbs in Jurkat human T-lymphoma cells.

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activity of the two anti-CD20 antibodies varied (Table 1). As shown in the Table 1, the cytotoxicity of TGLA was more effectively than rituximab. Analysis of ADCC in Daudi cells revealed that TGLA was the most cytotoxic, resulting in approximately 80% cell death at a concentration of 10 lg/mL, compared to approximately70% at 50 lg/ml for rituximab. The IC50 for TGLA ADCC cytotoxicity against Daudi cells was 0.84 ± 0.12 lg/mL (IC50 ± SD), and 6.43 ± 0.25 lg/mL for rituximab. Similar results were seen with Raji cells; the IC50 of TGLA and rituximab ADCC cytotoxicity against Raji cells were 0.69 ± 0.03 lg/mL and 4.72 ± 0.17 lg/ mL, respectively.

3.5. Serum stability The stability of the antibodies was important for the successful application in vivo. Therefore, we investigated the stability of TGLA stored in human serum at 37 °C. The data displayed in Fig. 4 shows that TGLA is highly stable in human serum, maintaining 80% binding activity after 10 days.

3.3. CDC activity of the anti-CD20 antibodies

3.6. Therapeutic efficacy

To explore the capacity of the anti-CD20 mAbs to mediate CDC, two CD20+ human lymphoma cell lines, Daudi and Raji, were used for this experiment, compared with T-lymphoma cell line Jurkat. TGLA and rituximab exhibited potent CDC activity. As with results observed in the proliferation evaluations, the levels of cytotoxicity were similar for rituximab and TGLA in this study (Fig. 3B).

To test in vivo activity of anti-CD20 antibodies, we inoculated Daudi cells subcutaneously into BALB/c nude mice on day 0. The mice were then treated with TGLA or rituximab, with human IgG as control. MAbs were administered on day 10, day 12 and day 14 at the dose of 5 mg/kg. The survival curves were plotted according to Kaplan–Meier method and compared using the log-rank test. As shown in Fig. 5A, both TGLA and rituximab were shown to significantly improve the survival of nude mice bearing Daudi tumors (P < 0.01 for each, compared with the human IgG control), and the activities of the two anti-CD20 antibodies in increasing survival were equal. The median survival time of control mice was 18.8 days after Daudi tumor inoculation. Median survival in the treated groups was extended to 25.7 days for rituximab and 26.2 days for TGLA. No obvious statistical difference was observed between the effects of TGLA and rituximab.

3.4. ADCC activity The anti-CD20 mAbs were compared for their ability to lyse CD20+ cells in the presence of PBMCs. The results (Fig. 3C) indicated that TGLA and rituximab were effective in inducing ADCC against Daudi or Raji cells in a dose-dependent manner, but not against Jurkat. Surprisingly, ADCC

Fig. 4. Serum stability of TGLA. TGLA was incubated in human serum at 37 °C for 3, 5, 7, or 10 days; binding activity was then determined by FACS. A, positive cells of TGLA binding to Daudi at different time points. B, the fluorescence intensity of TGLA binding to Daudi at different time points. C, the activity of TGLA at the given time points while the binding activity of the sample at time point zero was taken as 100%.

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Fig. 5. The in vivo anti-humor activity of anti-CD20 mAbs. A, The survival of tumor-bearing nude mice treated with anti-CD20 mAbs. Groups of 10 nude mice were inoculated subcutaneously with 3.5  106 Daudi cells on day 0, followed 10 d later by the intravenous injection of human IgG (j), rituximab (5) or TGLA (N). B, The volume of tumors treated with human IgG (j), rituximab (5) or TGLA (N); *, P < 0.05 mice treated with TGLA compared with rituximab; bars, SD. C, The weight of tumor-bearing mice treated with human IgG (j), rituximab (5) or TGLA (N); bar, SD. D, The toxicity of TGLA. Normal mice were injected with high doses of TGLA (500 lg); their weights were noted.

According to the equation, the tumor volumes were noted and calculated. Although all tumors eventually progressed, TGLA and rituximab therapies both significantly delayed tumor growth (Fig. 5B). Surprisingly, however, a pronounced difference was noticed between the two treatment groups: TGLA was significantly more effective in delaying tumor growth than rituximab. 3.7. TGLA toxicity Weight curves were plotted for in vivo therapy studies. The results showed that the weights of the mice in the studies were lightened significantly. Though no statistical difference was observed between the effects of human IgG control, TGLA and rituximab (Fig. 5C), we tested toxicity of the antibodies to eliminate their effect on the weight of mice. Normal mice were injected with high doses of TGLA (500 lg) and their weights were noted. The results characterized in Fig. 5D revealed the weights of mice injected with TGLA increased normally, similar results were achieved in in vivo studies in cavies (Data was not shown). These result indicated that TGLA didn’t affect survival and body weight in mice with the chosen dose.

4. Discussion It has been suggested that in vivo effector functions of therapeutic antibodies include ADCC and CDC, especially for the antibodies inducing anti-tumor activity; enhancement of these functions should be of therapeutic value [18,19]. Though the mechanisms of anti-CD20 antibody

therapies are still a matter of debate, CDC and ADCC are thought to be their predominant activities [9–12,20–22]. Several reports have shown CDC enhancement, both alone and in combination with ADCC enhancement, increases the antibodies’ anti-tumor activity and therapeutic capabilities [13–16]. In an attempt to improve anti-CD20 antibody therapeutic efficacy, we have developed a novel anti-CD20 antibody, TGLA. TGLA is a mouse/human chimeric antibody. Although a chimeric antibody is more likely than a humanized antibody to provoke an immune response, elicitation of a human antichimeric antibody response has not posed a significant obstacle to the use of anti-tumor mAbs. Though the immunogenicity of the chimeric antibody TGLA was theoretical and remains to be proven in clinical studies, such studies with the chimeric antibody rituximab have shown no serious immune responses in patients given rituximab [4–6,23]. TGLA is an engineered antibody based on a monoclonal antibody. To determine the binding activity of TGLA, several B-cell and T-cell lymphoma cell lines were used for immunofluorescence. The data described in Fig. 2A showed that TGLA binds to the B-cell lymphomas cell lines Raji and Daudi, but not to T-cell lymphoma cell lines Jurkat. To characterize TGLA binding to CD20 antigen further, TGLA

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was used to precipitate CD20 in Daudi cell lysate; the result shown in Fig. 2B confirms this. Data from the competitive binding assays shown that rituximab effectively competed with TGLA for binding to Daudi cells (Fig. 2C). These observations show that TGLA possesses specificity binding activity similar to rituximab, and uses the same epitope as rituximab. In this report, we used a panel of cell lines to evaluate the ability of the mAbs to kill NHL cells or inhibit them growth. TGLA was superior to rituximab in ADCC activities, though it was generally similar to rituximab in many in vitro activities such as CDC and inhibition of cell growth (Fig. 3). In the in vivo studies, though the activities of the two anti-CD20 antibodies in increasing survival were equal, TGLA was shown to be far more effective in delaying tumor growth than rituximab (Fig. 5). Because TGLA’s abilities to mediate CDC and inhibit cell growth against B-lymphoma cells were similar to those of rituximab, it could be concluded that the enhanced in vivo anti-tumor effect of TGLA is attributable to its marked increase in ADCC-inducing activity, although we were unable to discern the relative importance of this mechanism in improved therapeutic efficacy. Furthermore, TGLA was highly stable in human serum. TGLA could maintain 80% binding activity after 10 days’ storage in human serum at 37 °C (Fig. 4). A possible extension in serum half-life may permit extended dosing intervals and lead to reduced immunogenicity. Changes in pharmacokinetics and dosing regimens may affect the therapeutic response as well as toxicity [24,25]. The stability of TGLA would be likely to enhance its therapeutic response and reduce toxicity in vivo. In conclusion, the data shown here suggest that the mechanisms of cytotoxicity of TGLA, like rituximab, include inhibition of cell growth, as well as ADCC and CDC. Interesting, TGLA was superior to rituximab in ADCC activities, and far more effective in anti-tumor activity in vivo than rituximab. These findings suggest that the ADCC-enhanced anti-CD20 antibody TGLA might be an alternative therapeutic agent for B-cell lymphoma. It is expected that in humans, TGLA should be at least as effective as rituximab. Conflict of interest None declared. Acknowledgement This study was supported by the National ‘‘863” Fund (2006AA020503, 2007AA02Z306) National Sciences Fund (No. 30972807) of China. References [1] T.F. Tedder, P. Engel, CD20: a regulator of cell-cycle progression of B lymphocytes, Immunol Today 15 (1994) 450–454.

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[2] D.G. Maloney, Immunotherapy for non-Hodgkin’s lymphoma: monoclonal antibodies and vaccines, J Clin Oncol 23 (2005) 6421– 6428. [3] P. Martin, R.R. Furman, J. Ruan, R. Elstrom, J. Barrientos, R. Niesvizky, M. Coleman, Novel Leonard JP, lymphoma engineered anti-B-cell monoclonal antibodies for non-Hodgkin’s, Semin Hematol. 45 (2) (2008) 126–132. Apr. [4] M.J. Glennie, J.G. van de Winkel, Renaissance of cancer therapeutic antibodies, Drug Discov Today 8 (2003) 503–510. [5] G.A. Leget, M.S. Czuczman, Use of rituximab, the new FDA-approved antibody, Curr Opin Oncol 10 (1998) 548–551. [6] R.M. Sharkey, D.M. Goldenberg, Targeted therapy of cancer: new prospects for antibodies and immunoconjugates, CA Cancer J Clin 56 (2006) 226–243. [7] P. McLaughlin, A.J. Grillo-Lopez, B.K. Link, et al., Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program, J Clin Oncol 16 (1998) 2825–2833. [8] D.G. Maloney, A.J. Grillo-Lo´pez, D.J. Bodkin, et al., IDECC2B8: results of a phase I multiple-dose trial in patients with relapsed nonHodgkin’s lymphoma, J Clin Oncol 15 (1997) 3266–3274. [9] R. Marcus, A. Hagenbeek, The therapeutic use of rituximab in nonHodgkin’s lymphoma, Eur J Haematol Suppl. 67 (Jan) (2007) 5–14. [10] R.P. Taylor, M.A. Lindorfer, Immunotherapeutic mechanisms of antiCD20 monoclonal antibodies, Curr Opin Immunol. 20 (4) (2008) 444–449. Aug. [11] M.J. Glennie, R.R. French, M.S. Cragg, R.P. Taylor, Mechanism of killing by anti-CD20 monoclonal antibodies, Mol Immunol 44 (2007) 3823–3837. [12] J. Golay, R. Gramigna, V. Facchinetti, D. Capello, M. Introna, Acquired immunodeficiency syndrome-associated lymphomas are efficiently lysed through complement-dependent cytotoxicity and antibodydependent cellular cytotoxicity by rituximab, Br J Haematol 119 (2002) 923–929. [13] Natsume A, Shimizu-Yokoyama Y, Satoh M, Shitara K, Niwa R.Engineered anti-CD20 antibodies with enhanced complementactivating capacity mediate potent anti-lymphoma activity. Cancer Sci. 2009 Aug 25. [Epub ahead of print]. [14] M. Nishida, S. Usuda, M. Okabe, H. Miyakoda, M. Komatsu, H. Hanaoka, K. Teshigawara, O. Niwa, Characterization of novel murine anti-CD20 monoclonal antibodies and their comparison to 2B8 and c2B8 (rituximab), Int J Oncol. 31 (1) (2007) 29–40. Jul. [15] B. Li, S. Shi, W. Qian, L. Zhao, D. Zhang, S. Hou, L. Zheng, J. Dai, J. Zhao, H. Wang, Y. Guo, Development of novel tetravalent anti-CD20 antibodies with potent antitumor activity, Cancer Res. 68 (7) (2008) 2400–2408. Apr 1. [16] E.A. Rossi, D.M. Goldenberg, T.M. Cardillo, R. Stein, Y. Wang, C.H. Chang, Novel designs of multivalent anti-CD20 humanized antibodies as improved lymphoma therapeutics, Cancer Res. 68 (20) (2008) 8384–8392. Oct 15. [17] Y. Huang, Y. Li, Y.G. Wang, X. Gu, Y. Wang, B.F. Shen, An efficient and targeted gene integration system for high-level antibody expression, J Immunol Methods. 322 (1-2) (2007) 28–39. Apr 30. [18] R.A. Clynes, T.L. Towers, L.G. Presta, J.V. Ravetch, Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets, Nat Med 6 (2000) 443–446. [19] N. Di Gaetano, E. Cittera, R. Nota, et al., Complement activation determines the therapeutic activity of rituximab in vivo, J Immunol 171 (2003) 1581–1587. [20] A.D. Kennedy, P.V. Beum, M.D. Solga, et al., Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia, J Immunol 172 (2004) 3280–3288. [21] G. Cartron, L. Dacheux, G. Salles, et al., Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgRIIIa gene, Blood 99 (2002) 754–758. [22] W.K. Weng, R. Levy, Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma, J Clin Oncol 21 (2003) 3940–3947. [23] Therapy with immunoglobulin: applications for monoclonal antibodies C.P. Stowell, J Infus Nurs. 29 (3 Suppl) (2006) S29–44. May-Jun. [24] Rituximab: a review of its use in non-Hodgkin’s lymphoma and chronic lymphocytic leukaemia G.L. Plosker, D.P. Figgitt, Drugs. 63 (8) (2003) 803–843. [25] Immunologic monitoring during OKT 3 therapy L. Chatenoud, Clin Transplant. 7 (4 Pt 2) (1993) 422–430. Aug.