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mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab abc
Lei Zhao
b
abde
, Feiyue Xie , Xin Tong d
d
a
a
a
ac
Juan Zheng , Ziye Zhao , Bohua Li , Dapeng Zhang , Jian Zhao abcd
Tong
abcd
, Sheng Hou
a
d
, Huafei Li , Yaling Chen , Weizhu Qian , Shuyan Duan , abc
abcd
, Jianxin Dai
, Xin
abcd
& Yajun Guo
a
International Joint Cancer Institute; Second Military Medical University; Shanghai, PR China
b
PLA General Hospital Cancer Center; PLA School of Medicine; Beijing, PR China
c
The State Key Laboratory of Antibody Medicine & Targeting Therapy and Shanghai Key Laboratory of Cell Engineering & Antibody; Shanghai, PR China d
Click for updates
School of Pharmaceutical; Liaocheng University; Liaocheng, PR China
e
These authors contributed equally to this work. Published online: 26 Mar 2014.
To cite this article: Lei Zhao, Feiyue Xie, Xin Tong, Huafei Li, Yaling Chen, Weizhu Qian, Shuyan Duan, Juan Zheng, Ziye Zhao, Bohua Li, Dapeng Zhang, Jian Zhao, Jianxin Dai, Xin Tong, Sheng Hou & Yajun Guo (2014) Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab, mAbs, 6:3, 739-747, DOI: 10.4161/mabs.28613 To link to this article: http://dx.doi.org/10.4161/mabs.28613
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
report
Paper Type
mAbs 6:3, 739–747; May/June 2014; © 2014 Landes Bioscience
Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab Lei Zhao1,2,3,†, Feiyue Xie2,†, Xin Tong1,2,4,†, Huafei Li1, Yaling Chen1, Weizhu Qian1, Shuyan Duan4, Juan Zheng4, Ziye Zhao4, Bohua Li1, Dapeng Zhang1,3, Jian Zhao1,2,3, Jianxin Dai1,2,3,4, Hao Wang1,2,3,4, Sheng Hou1,2,3,4,* and Yajun Guo1,2,3,4,* International Joint Cancer Institute; Second Military Medical University; Shanghai, PR China; 2PLA General Hospital Cancer Center; PLA School of Medicine; Beijing, PR China; 3The State Key Laboratory of Antibody Medicine & Targeting Therapy and Shanghai Key Laboratory of Cell Engineering & Antibody; Shanghai, PR China; 4 School of Pharmaceutical; Liaocheng University; Liaocheng, PR China
1
These authors contributed equally to this work.
Downloaded by [University of Saskatchewan Library] at 10:27 16 March 2015
†
Keywords: CD20 antibody, rituximab, HLA-DR, lysosome-mediated cell death, CrossMab, resistant cancer cell
Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cellmediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.
Introduction Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignancies that represents approximately 4% of all cancers. More than 90% of NHLs have a B-cell phenotype, and almost all express cell surface CD20, a B cell-specific member of the MS4A gene family.1,2 Rituximab, the first monoclonal antibody (mAb) approved for cancer treatment, has revolutionized the management and treatment of B-cell malignancies, increasing the median overall survival of patients with many of these diseases.3,4 Despite widespread use of rituximab, the efficacy remains variable and often modest, and the pursuit of improved agents to replace rituximab is intense, with several candidates currently under clinical evaluation.5,6 Most have been selected and engineered to provide a range of potential advantages, including increased binding avidity, reduced immunogenicity, enhanced direct cell death as mediated by type II CD20 antibodies and improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).3,7-10 However, complex diseases are often multifactorial in nature, and
involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks.11,12 Thus, simultaneous blockade of these effector molecules is likely to provide better clinical efficacy and/ or reach a broader patient population than inhibition of a single target.13 Previous studies indicated that blockade of multiple targets, which can be achieved by the combination of several mAbs or bispecific antibodies generated through biochemical or genetic approaches, results in improved therapeutic efficacy.14,15 However, combination therapy of several mAbs increases healthcare costs and the financial burden to families and societies, and the option of using several approved mAbs for combination therapy is limited because of the small number of therapeutic mAbs currently on the market. These data show the urgent need to design bispecific antibody with potent anti-tumor activities against NHL. Acquired therapy resistance is one of the prime obstacles for successful cancer treatment. Resistance is often acquired already during an early phase of tumor development when genetic changes cause defects in caspase-dependent apoptosis
*Correspondence to: Sheng Hou; Email: [email protected]; Yajun Guo; Email: [email protected] Submitted: 02/07/2014; Revised: 03/17/2014; Accepted: 03/20/2014; Published Online: 03/26/2014 http://dx.doi.org/10.4161/mabs.28613 www.landesbioscience.com mAbs
739
Downloaded by [University of Saskatchewan Library] at 10:27 16 March 2015
safety concerns have been raised regarding the clinical use of HLA-DR-directed antibodies. To reduce reliance on intact immunologic systems in the patient and effector mechanism-related toxicity, Goldenberg and his colleagues have replaced the Fc region of hL243γ1, the humanized IgG1 anti-HLA-DR mAb L243, with the IgG4 isotope (IMMU-114) to abrogate the effector cell functions of the antibody (ADCC and CDC). Their data indicated that anti-HLA-DR antibody hL243γ1 could not only induce potent lysosome-mediated cell death against lymphoma and leukemia cells, but also significantly increase anti-proliferative activity after combination with rituximab.23 While acknowledging that HLA-DR is not a tumor-specific antigen, HLA-DR remains an attractive molecule with potential as a target for a CD20-HLA-DR bispecific antibody. To our knowledge, the classical IgG architecture, as it Figure 1. Characterization of CD20–243 CrossMab. (A) Schematic diagram of was selected during evolution, has many advantages for the Fab domain exchange resulting in the generation of CD20–243 bispecific the therapeutic application of bispecific antibodies.24,25 antibody when combined with the KiH technology. (B and C) Binding of The Fc part is identical to that of a conventional IgG 125 I-labeled CD20–243 CrossMab, rituximab Fab, hL243γ1 Fab and IMMU-114 Fab to antibody, resulting in IgG-like pharmacokinetic CHO-CD20 cells (B) or Nalmalwa (C). 125I-labeled CD20-Flex BiFP and other mAbs properties and retained effector functions such as the were separately incubated with CHO-CD20 cells for 2 h at 37 °C. The saturation of CD20–243 CrossMab is approximately 10μg/ml, which is comparable to rituximab. mediation of ADCC through FcγRIIIa binding. IgGThe cell-bound and free 125I-labeled CrossMab or mAbs were then separated by like size and molecular weight are expected to result in centrifugation through phthalate oils and the cell pellets together with bound IgG-like diffusion, tumor penetration, and accumulation antibody counted for radioactivity. Data from saturation binding experiments in comparison with bispecific tetravalent antibodies of were analyzed by nonlinear least-squares regression for curve-fitting and Kd higher molecular weight. Considering these benefits, we estimation. Data are mean ± SD (n = 3). (D) Dissociation of 125I-labeled CD20–243 CrossMab, rituximab, hL243γ1 and IMMU-114 from Raji cells. Cells were incubated converted the CD20 antibody rituximab and HLA-DR with 125I-labeled CD20–243 CrossMab, rituximab or hL243γ1 and IMMU-114 (10 μg/ antibody hL243γ1 into an IgG-like bispecific antibody ml) at 37 °C for 1 h, washed twice, and resuspended. Samples of cells were taken (CD20–243 CrossMab) by using CrossMab technology.26 at time 0, 1, 2 and 4 h and then washed and analyzed. Shown are means and SD of Our results indicated that CD20–243 CrossMab induces at least three experiments. significantly high levels of CDC, ADCC and cell death in NHL, and has potent anti-tumor capacities against both pathways and provide transformed cells with higher growth B-lymphoma cells and rituximab-resistant (RR) B-lymphoma and survival potential.11,12,16 Additionally, cancers treated with cells. More importantly, although HLA-DR is not specifically chemotherapeutic drugs often acquire the ability to efflux drugs expressed on malignant cells, CD20–243 CrossMab exhibits by increasing the expression of multidrug resistance (MDR) specific anti-tumor activities against both CD20 and HLA-DR proteins, P-glycoproteins of the ATP-binding cassette transporter positive malignant cells. family.17 Thus, alternative cell death pathways capable of killing apoptosis- and therapy-resistant cancer cells have attracted vast interest among cancer researchers. Growing evidence indicated Results that lysosomes can be considered as an “Achilles heel” for Design and characterization of CD20–243 CrossMab selectively destroying cancer cells, which has been demonstrated Based on CrossMab technology recently reported,26 we as an effective way to kill apoptosis-resistant cancer cells and 18,19 Recently, designed an IgG-like bispecific CrossMab (CD20–243 CrossMab) re-sensitize MDR cells to classical chemotherapy. Ivanov and colleagues have revealed that, although lysosome- that deviates only minimally from the naturally occurring CD20 mediated cell death can be elicited by both type II CD20 mAbs antibody rituximab and HLA-DR antibody hL243γ1. As shown and HLA-DR antibody L243, HLA-DR antibody L243 could in the right of Figure 1A, the constant heavy 1 (CH1) of hL243γ1 induce more potent lysosome-mediated cell death than type II was replaced with the antibody constant light (CL), generating CD20 mAbs, suggesting that HLA-DR can be used as an ideal a polypeptide chain made of hL243 HV-CL-Hinge-CH2 and target for induction of lysosome-mediated cell death against CH3. To generate CD20–243 CrossMab, exchange of CH1 lymphoma.20 Previous studies demonstrated that, although and CL domains of hL243γ1 is essential for correct association HLA-DR is expressed at high levels on a range of hematologic of the light chain and the cognate heavy chain of the half IgG malignancies, it is constitutively expressed on normal B cells, of rituximab in CD20–243 CrossMab. Hetero-dimerization monocytes/macrophages and dendritic cells.21,22 Due to the fact of the heavy chain of rituximab and hL243γ1 was achieved by that the antigen is expressed on normal as well as tumor cells, using the “knobs into holes” (KiH) method.27,28 The resulting
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highly purified CD20–243 CrossMab was assessed on SDS/PAGE (Fig. S1A) and size-exclusion HPLC (Fig. S1B). Then, we used CHO-CD20 (high expression of CD20), Namalwa (high expression of HLA-DR) and Raji (high expression of both CD20 and HLA-DR) cells to determine the binding activity of CrossMab. The binding affinity for CD20 and HLA-DR of the CD20–243 CrossMab was determined by analyzing direct cell surface saturation binding to CHO-CD20 cells (Fig. 1B) and Namalwa cells (Fig. 1c), respectively. The dissociation constant of rituximab (4.83 ± 0.32 nM) is quantitatively consistent with a previous report by Reff et al.29 It can be seen in Figure 1B that the Figure 2. The CDC and ADCC activities of CD20–243 CrossMab against lymphoma. Raji (A), CHO-CD20 CD20–243 CrossMab binding affinity (B) and Namalwa (C) cells were incubated with increasing concentrations of CD20–243 CrossMab or for CD20 (4.79 ± 0.28 nM) is similar other mAbs in the presence of human complement at 37 °C for 4 h. CDC activity was calculated by a standard LDH assay as described in “Materials and methods.” These graphs are representative of at to the affinity of rituximab. In addition, least 3 experiments, each showing similar results. (D–F) ADCC activity against Raji (D), CHO-CD20 (E) the binding affinity of hL243γ1 and and Namalwa (F) cells using human PBMCs as effector cells at E:T ratio of 25:1. The ADCC activity of CD20–243 CrossMab for HLA-DR CD20–243 CrossMab or other mAbs at varying concentrations was measured using a standard LDH is very similar (Fig. 1C). As shown in assay as described in “Materials and Methods.” Data are expressed as means ± SD (n = 3). Figure S1C, CrossMab could bind to CD20 and HLA-DR positive Raji cell healthy donors were used as effector cells and Daudi cells were used with similar high level as that of IMMU-114 and hL243γ1. Binding “off-rate” experiments using 125I-labeled IgG were as the target. Assays were conducted at effector:target (E:T) cell performed to compare the dissociation of rituximab and CD20– ratios of 25:1 using antibody concentrations ranging from 0.003 243 CrossMab from Raji cells. As shown in Figure 1D, the data to 10 μg/ml (Fig. 2D–F). Our data showed that approximately showed a slight difference in the off-rate between CD20–243 2 μg/ml of rituximab or CrossMab was required to achieve the CrossMab and rituximab. Approximately 50% of the rituximab, 50% targeted lysis levels at E:T ratios of 25:1. Furthermore, and more than 40% of CD20–243 CrossMab, remained bound ADCC activity of CD20–243 CrossMab on CHO-CD20 cells to the cells after 4 h. In addition, similar results were achieved and Namalwa cells were analyzed (Fig. 2E and F). In line with with F(ab)2, which excluded an interaction with FcγR on target our expectation, significant decreases of ADCC activity against cells influencing mAb dissociation (data not shown). These CHO-CD20 cells and Namalwa cell were observed after treatment data indicated that CD20–243 CrossMab nearly kept the intact with CrossMab. These data indicated that CD20–243 CrossMab affinity bound to CD20 and HLA-DR, and exhibited similar with specific anti-tumor activities can induce remarkable CDC and ADCC against both CD20 and HLA-DR positive lymphoma binding avidity against B-cell lymphoma. CD20–243 CrossMab with specific CDC and ADCC cells. Unusually potent antiproliferative activity of CD20–243 activities efficiently eradicate B-cell lymphoma comparable to CrossMab rituximab In clinical studies, the facts that (1) responding patients The cytotoxic activities of CD20–243 CrossMab were first assessed against Raji cells. In line with our expectations, CrossMab generally displayed progressive tumor mass reduction, and displayed approximately the same high level of CDC activity as (2) complete response is usually achieved several weeks after rituximab (Fig. 2A). To further investigate the specific cytotoxic completion of therapy, suggest that, in vivo, cell growth activities of CrossMab, CHO-CD20 cells or Namalwa cells that inhibition could play an important role in cancer treatment.30 expressed a high level of CD20 or HLA-DR proteins, respectively, Thus, the effect of CD20–243 CrossMab on cellular proliferation were used in our further studies. Compared with rituximab or was assessed using the 3H-thymidine uptake assay on Ramos, hL243γ1, the cytotoxic activities of CD20–243 CrossMab on CHO-CD20, and Namalwa cells, separately. Unexpectedly, our CHO-CD20 cells and Namalwa cells were significantly decreased, data showed that, after treatment with CD20–243 CrossMab, separately (Fig. 2B and C). These results suggested that CD20– the significant inhibition of proliferation was observed (Fig. 3A). 243 CrossMab could trigger specific cytotoxic activities against Although hL243γ1 or the combination of rituximab and B-cell lymphoma. Then, a standard LDH assay was performed to hL243γ1 exhibited potent anti-proliferative capacity after determine the CrossMab-mediated ADCC by human peripheral cross-linking with second antibody, CD20–243 CrossMab still blood mononuclear cells (PBMCs). Purified human PBMCs from displayed high level of anti-proliferative activity even without
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Figure 3. Anti-proliferative effects of CD20–243 CrossMab. Effects of CrossMab on proliferation of Raji (A), CHO-CD20 (B) and Namalwa (C) cells were determined by 3H-thymidine uptake assays. Cells were cultured with CrossMab or other mAbs with or without a second antibody for cross-linking. Error bars represent SD of triplicates.
cross-linking (Fig. 3A). Then, the anti-proliferative activity against CHO-CD20 or Namalwa cells was explored. Our data clearly showed that, even with very low expression of CD20 on Namalwa cells, CD20–243 CrossMab showed potent antiproliferative activity against malignancies (Fig. 3B and C). Induction of Lysosome-mediated cell death by CD20–243 CrossMab Growing evidence has revealed that lysosomes are excellent pharmacological targets for selectively destroying cancer cells, and this approach recently emerged as an effective way to kill apoptosis resistant cancer cells and re-sensitize MDR cells to classical chemotherapy.18,19 To investigate the cell death induced by CD20–243 CrossMab, pan-caspase inhibitor was first used in our experiments. As indicated in Figure 4A, although rituximab triggered a low level of cell death (
mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab abc
Lei Zhao
b
abde
, Feiyue Xie , Xin Tong d
d
a
a
a
ac
Juan Zheng , Ziye Zhao , Bohua Li , Dapeng Zhang , Jian Zhao abcd
Tong
abcd
, Sheng Hou
a
d
, Huafei Li , Yaling Chen , Weizhu Qian , Shuyan Duan , abc
abcd
, Jianxin Dai
, Xin
abcd
& Yajun Guo
a
International Joint Cancer Institute; Second Military Medical University; Shanghai, PR China
b
PLA General Hospital Cancer Center; PLA School of Medicine; Beijing, PR China
c
The State Key Laboratory of Antibody Medicine & Targeting Therapy and Shanghai Key Laboratory of Cell Engineering & Antibody; Shanghai, PR China d
Click for updates
School of Pharmaceutical; Liaocheng University; Liaocheng, PR China
e
These authors contributed equally to this work. Published online: 26 Mar 2014.
To cite this article: Lei Zhao, Feiyue Xie, Xin Tong, Huafei Li, Yaling Chen, Weizhu Qian, Shuyan Duan, Juan Zheng, Ziye Zhao, Bohua Li, Dapeng Zhang, Jian Zhao, Jianxin Dai, Xin Tong, Sheng Hou & Yajun Guo (2014) Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab, mAbs, 6:3, 739-747, DOI: 10.4161/mabs.28613 To link to this article: http://dx.doi.org/10.4161/mabs.28613
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
report
Paper Type
mAbs 6:3, 739–747; May/June 2014; © 2014 Landes Bioscience
Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab Lei Zhao1,2,3,†, Feiyue Xie2,†, Xin Tong1,2,4,†, Huafei Li1, Yaling Chen1, Weizhu Qian1, Shuyan Duan4, Juan Zheng4, Ziye Zhao4, Bohua Li1, Dapeng Zhang1,3, Jian Zhao1,2,3, Jianxin Dai1,2,3,4, Hao Wang1,2,3,4, Sheng Hou1,2,3,4,* and Yajun Guo1,2,3,4,* International Joint Cancer Institute; Second Military Medical University; Shanghai, PR China; 2PLA General Hospital Cancer Center; PLA School of Medicine; Beijing, PR China; 3The State Key Laboratory of Antibody Medicine & Targeting Therapy and Shanghai Key Laboratory of Cell Engineering & Antibody; Shanghai, PR China; 4 School of Pharmaceutical; Liaocheng University; Liaocheng, PR China
1
These authors contributed equally to this work.
Downloaded by [University of Saskatchewan Library] at 10:27 16 March 2015
†
Keywords: CD20 antibody, rituximab, HLA-DR, lysosome-mediated cell death, CrossMab, resistant cancer cell
Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cellmediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.
Introduction Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignancies that represents approximately 4% of all cancers. More than 90% of NHLs have a B-cell phenotype, and almost all express cell surface CD20, a B cell-specific member of the MS4A gene family.1,2 Rituximab, the first monoclonal antibody (mAb) approved for cancer treatment, has revolutionized the management and treatment of B-cell malignancies, increasing the median overall survival of patients with many of these diseases.3,4 Despite widespread use of rituximab, the efficacy remains variable and often modest, and the pursuit of improved agents to replace rituximab is intense, with several candidates currently under clinical evaluation.5,6 Most have been selected and engineered to provide a range of potential advantages, including increased binding avidity, reduced immunogenicity, enhanced direct cell death as mediated by type II CD20 antibodies and improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).3,7-10 However, complex diseases are often multifactorial in nature, and
involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks.11,12 Thus, simultaneous blockade of these effector molecules is likely to provide better clinical efficacy and/ or reach a broader patient population than inhibition of a single target.13 Previous studies indicated that blockade of multiple targets, which can be achieved by the combination of several mAbs or bispecific antibodies generated through biochemical or genetic approaches, results in improved therapeutic efficacy.14,15 However, combination therapy of several mAbs increases healthcare costs and the financial burden to families and societies, and the option of using several approved mAbs for combination therapy is limited because of the small number of therapeutic mAbs currently on the market. These data show the urgent need to design bispecific antibody with potent anti-tumor activities against NHL. Acquired therapy resistance is one of the prime obstacles for successful cancer treatment. Resistance is often acquired already during an early phase of tumor development when genetic changes cause defects in caspase-dependent apoptosis
*Correspondence to: Sheng Hou; Email: [email protected]; Yajun Guo; Email: [email protected] Submitted: 02/07/2014; Revised: 03/17/2014; Accepted: 03/20/2014; Published Online: 03/26/2014 http://dx.doi.org/10.4161/mabs.28613 www.landesbioscience.com mAbs
739
Downloaded by [University of Saskatchewan Library] at 10:27 16 March 2015
safety concerns have been raised regarding the clinical use of HLA-DR-directed antibodies. To reduce reliance on intact immunologic systems in the patient and effector mechanism-related toxicity, Goldenberg and his colleagues have replaced the Fc region of hL243γ1, the humanized IgG1 anti-HLA-DR mAb L243, with the IgG4 isotope (IMMU-114) to abrogate the effector cell functions of the antibody (ADCC and CDC). Their data indicated that anti-HLA-DR antibody hL243γ1 could not only induce potent lysosome-mediated cell death against lymphoma and leukemia cells, but also significantly increase anti-proliferative activity after combination with rituximab.23 While acknowledging that HLA-DR is not a tumor-specific antigen, HLA-DR remains an attractive molecule with potential as a target for a CD20-HLA-DR bispecific antibody. To our knowledge, the classical IgG architecture, as it Figure 1. Characterization of CD20–243 CrossMab. (A) Schematic diagram of was selected during evolution, has many advantages for the Fab domain exchange resulting in the generation of CD20–243 bispecific the therapeutic application of bispecific antibodies.24,25 antibody when combined with the KiH technology. (B and C) Binding of The Fc part is identical to that of a conventional IgG 125 I-labeled CD20–243 CrossMab, rituximab Fab, hL243γ1 Fab and IMMU-114 Fab to antibody, resulting in IgG-like pharmacokinetic CHO-CD20 cells (B) or Nalmalwa (C). 125I-labeled CD20-Flex BiFP and other mAbs properties and retained effector functions such as the were separately incubated with CHO-CD20 cells for 2 h at 37 °C. The saturation of CD20–243 CrossMab is approximately 10μg/ml, which is comparable to rituximab. mediation of ADCC through FcγRIIIa binding. IgGThe cell-bound and free 125I-labeled CrossMab or mAbs were then separated by like size and molecular weight are expected to result in centrifugation through phthalate oils and the cell pellets together with bound IgG-like diffusion, tumor penetration, and accumulation antibody counted for radioactivity. Data from saturation binding experiments in comparison with bispecific tetravalent antibodies of were analyzed by nonlinear least-squares regression for curve-fitting and Kd higher molecular weight. Considering these benefits, we estimation. Data are mean ± SD (n = 3). (D) Dissociation of 125I-labeled CD20–243 CrossMab, rituximab, hL243γ1 and IMMU-114 from Raji cells. Cells were incubated converted the CD20 antibody rituximab and HLA-DR with 125I-labeled CD20–243 CrossMab, rituximab or hL243γ1 and IMMU-114 (10 μg/ antibody hL243γ1 into an IgG-like bispecific antibody ml) at 37 °C for 1 h, washed twice, and resuspended. Samples of cells were taken (CD20–243 CrossMab) by using CrossMab technology.26 at time 0, 1, 2 and 4 h and then washed and analyzed. Shown are means and SD of Our results indicated that CD20–243 CrossMab induces at least three experiments. significantly high levels of CDC, ADCC and cell death in NHL, and has potent anti-tumor capacities against both pathways and provide transformed cells with higher growth B-lymphoma cells and rituximab-resistant (RR) B-lymphoma and survival potential.11,12,16 Additionally, cancers treated with cells. More importantly, although HLA-DR is not specifically chemotherapeutic drugs often acquire the ability to efflux drugs expressed on malignant cells, CD20–243 CrossMab exhibits by increasing the expression of multidrug resistance (MDR) specific anti-tumor activities against both CD20 and HLA-DR proteins, P-glycoproteins of the ATP-binding cassette transporter positive malignant cells. family.17 Thus, alternative cell death pathways capable of killing apoptosis- and therapy-resistant cancer cells have attracted vast interest among cancer researchers. Growing evidence indicated Results that lysosomes can be considered as an “Achilles heel” for Design and characterization of CD20–243 CrossMab selectively destroying cancer cells, which has been demonstrated Based on CrossMab technology recently reported,26 we as an effective way to kill apoptosis-resistant cancer cells and 18,19 Recently, designed an IgG-like bispecific CrossMab (CD20–243 CrossMab) re-sensitize MDR cells to classical chemotherapy. Ivanov and colleagues have revealed that, although lysosome- that deviates only minimally from the naturally occurring CD20 mediated cell death can be elicited by both type II CD20 mAbs antibody rituximab and HLA-DR antibody hL243γ1. As shown and HLA-DR antibody L243, HLA-DR antibody L243 could in the right of Figure 1A, the constant heavy 1 (CH1) of hL243γ1 induce more potent lysosome-mediated cell death than type II was replaced with the antibody constant light (CL), generating CD20 mAbs, suggesting that HLA-DR can be used as an ideal a polypeptide chain made of hL243 HV-CL-Hinge-CH2 and target for induction of lysosome-mediated cell death against CH3. To generate CD20–243 CrossMab, exchange of CH1 lymphoma.20 Previous studies demonstrated that, although and CL domains of hL243γ1 is essential for correct association HLA-DR is expressed at high levels on a range of hematologic of the light chain and the cognate heavy chain of the half IgG malignancies, it is constitutively expressed on normal B cells, of rituximab in CD20–243 CrossMab. Hetero-dimerization monocytes/macrophages and dendritic cells.21,22 Due to the fact of the heavy chain of rituximab and hL243γ1 was achieved by that the antigen is expressed on normal as well as tumor cells, using the “knobs into holes” (KiH) method.27,28 The resulting
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Volume 6 Issue 3
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highly purified CD20–243 CrossMab was assessed on SDS/PAGE (Fig. S1A) and size-exclusion HPLC (Fig. S1B). Then, we used CHO-CD20 (high expression of CD20), Namalwa (high expression of HLA-DR) and Raji (high expression of both CD20 and HLA-DR) cells to determine the binding activity of CrossMab. The binding affinity for CD20 and HLA-DR of the CD20–243 CrossMab was determined by analyzing direct cell surface saturation binding to CHO-CD20 cells (Fig. 1B) and Namalwa cells (Fig. 1c), respectively. The dissociation constant of rituximab (4.83 ± 0.32 nM) is quantitatively consistent with a previous report by Reff et al.29 It can be seen in Figure 1B that the Figure 2. The CDC and ADCC activities of CD20–243 CrossMab against lymphoma. Raji (A), CHO-CD20 CD20–243 CrossMab binding affinity (B) and Namalwa (C) cells were incubated with increasing concentrations of CD20–243 CrossMab or for CD20 (4.79 ± 0.28 nM) is similar other mAbs in the presence of human complement at 37 °C for 4 h. CDC activity was calculated by a standard LDH assay as described in “Materials and methods.” These graphs are representative of at to the affinity of rituximab. In addition, least 3 experiments, each showing similar results. (D–F) ADCC activity against Raji (D), CHO-CD20 (E) the binding affinity of hL243γ1 and and Namalwa (F) cells using human PBMCs as effector cells at E:T ratio of 25:1. The ADCC activity of CD20–243 CrossMab for HLA-DR CD20–243 CrossMab or other mAbs at varying concentrations was measured using a standard LDH is very similar (Fig. 1C). As shown in assay as described in “Materials and Methods.” Data are expressed as means ± SD (n = 3). Figure S1C, CrossMab could bind to CD20 and HLA-DR positive Raji cell healthy donors were used as effector cells and Daudi cells were used with similar high level as that of IMMU-114 and hL243γ1. Binding “off-rate” experiments using 125I-labeled IgG were as the target. Assays were conducted at effector:target (E:T) cell performed to compare the dissociation of rituximab and CD20– ratios of 25:1 using antibody concentrations ranging from 0.003 243 CrossMab from Raji cells. As shown in Figure 1D, the data to 10 μg/ml (Fig. 2D–F). Our data showed that approximately showed a slight difference in the off-rate between CD20–243 2 μg/ml of rituximab or CrossMab was required to achieve the CrossMab and rituximab. Approximately 50% of the rituximab, 50% targeted lysis levels at E:T ratios of 25:1. Furthermore, and more than 40% of CD20–243 CrossMab, remained bound ADCC activity of CD20–243 CrossMab on CHO-CD20 cells to the cells after 4 h. In addition, similar results were achieved and Namalwa cells were analyzed (Fig. 2E and F). In line with with F(ab)2, which excluded an interaction with FcγR on target our expectation, significant decreases of ADCC activity against cells influencing mAb dissociation (data not shown). These CHO-CD20 cells and Namalwa cell were observed after treatment data indicated that CD20–243 CrossMab nearly kept the intact with CrossMab. These data indicated that CD20–243 CrossMab affinity bound to CD20 and HLA-DR, and exhibited similar with specific anti-tumor activities can induce remarkable CDC and ADCC against both CD20 and HLA-DR positive lymphoma binding avidity against B-cell lymphoma. CD20–243 CrossMab with specific CDC and ADCC cells. Unusually potent antiproliferative activity of CD20–243 activities efficiently eradicate B-cell lymphoma comparable to CrossMab rituximab In clinical studies, the facts that (1) responding patients The cytotoxic activities of CD20–243 CrossMab were first assessed against Raji cells. In line with our expectations, CrossMab generally displayed progressive tumor mass reduction, and displayed approximately the same high level of CDC activity as (2) complete response is usually achieved several weeks after rituximab (Fig. 2A). To further investigate the specific cytotoxic completion of therapy, suggest that, in vivo, cell growth activities of CrossMab, CHO-CD20 cells or Namalwa cells that inhibition could play an important role in cancer treatment.30 expressed a high level of CD20 or HLA-DR proteins, respectively, Thus, the effect of CD20–243 CrossMab on cellular proliferation were used in our further studies. Compared with rituximab or was assessed using the 3H-thymidine uptake assay on Ramos, hL243γ1, the cytotoxic activities of CD20–243 CrossMab on CHO-CD20, and Namalwa cells, separately. Unexpectedly, our CHO-CD20 cells and Namalwa cells were significantly decreased, data showed that, after treatment with CD20–243 CrossMab, separately (Fig. 2B and C). These results suggested that CD20– the significant inhibition of proliferation was observed (Fig. 3A). 243 CrossMab could trigger specific cytotoxic activities against Although hL243γ1 or the combination of rituximab and B-cell lymphoma. Then, a standard LDH assay was performed to hL243γ1 exhibited potent anti-proliferative capacity after determine the CrossMab-mediated ADCC by human peripheral cross-linking with second antibody, CD20–243 CrossMab still blood mononuclear cells (PBMCs). Purified human PBMCs from displayed high level of anti-proliferative activity even without
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Figure 3. Anti-proliferative effects of CD20–243 CrossMab. Effects of CrossMab on proliferation of Raji (A), CHO-CD20 (B) and Namalwa (C) cells were determined by 3H-thymidine uptake assays. Cells were cultured with CrossMab or other mAbs with or without a second antibody for cross-linking. Error bars represent SD of triplicates.
cross-linking (Fig. 3A). Then, the anti-proliferative activity against CHO-CD20 or Namalwa cells was explored. Our data clearly showed that, even with very low expression of CD20 on Namalwa cells, CD20–243 CrossMab showed potent antiproliferative activity against malignancies (Fig. 3B and C). Induction of Lysosome-mediated cell death by CD20–243 CrossMab Growing evidence has revealed that lysosomes are excellent pharmacological targets for selectively destroying cancer cells, and this approach recently emerged as an effective way to kill apoptosis resistant cancer cells and re-sensitize MDR cells to classical chemotherapy.18,19 To investigate the cell death induced by CD20–243 CrossMab, pan-caspase inhibitor was first used in our experiments. As indicated in Figure 4A, although rituximab triggered a low level of cell death (
