J Cancer Res Clin Oncol DOI 10.1007/s00432-015-1949-7
ORIGINAL ARTICLE – CANCER RESEARCH
A bispecific antibody directly induces lymphoma cell death by simultaneously targeting CD20 and HLA‑DR Jing Zeng · Ran Liu · Jinjing Wang · Yi Fang
Received: 13 July 2014 / Accepted: 23 February 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose Both CD20 and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas and are therapeutic targets in antibody-based lymphoma therapy. The aim of this study was to evaluate the anti-tumor effect of a bispecific antibody CD20-HLA-DR DVD-Ig on B-cell lymphoma. Methods The gene for bispecific antibody CD20HLA-DR DVD-Ig was constructed and expressed in FreeStyle™293-F cells, followed by purification. Their functions were characterized for binding to CD20 and HLA-DR and for cytotoxicity against B-cell lymphoma. Results The bispecific antibody CD20-HLA-DR DVDIg was engineered using the DNA fragments for the antiCD20 rituximab and anti-HLA-DR hL243γ1. The CD20HLA-DR DVD-Ig bound simultaneously to both CD20 and HLA-DR, induced potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against B-cell lymphoma, and elicited homotypic adhesion and actin reorganization. Treatment of a mixture of human whole blood and Raji cells with CD20-HLA-DR
Jing Zeng contributed firstly to this work. J. Zeng · R. Liu · J. Wang · Y. Fang (*) Endocrinology Department, Affiliated Hospital of the Academy of Military Medical Sciences, 8 Dongdajie, Beijing 100071, People’s Republic of China e-mail: [email protected] J. Zeng e-mail: [email protected] J. Zeng Cancer Center, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China
DVD-Ig effectively depleted Raji cells and had a little toxicity against normal B cells. Conclusion Our data indicated that targeting both CD20 and HLA-DR was an effective way against NHL, suggesting that CD20-HLA-DR DVD-Ig may be a promising therapeutic agent for B-cell lymphoma. Keywords Bispecific antibody · Cell death · CD20 · HLA-DR · Lymphoma
Introduction Although traditional approaches for treatment of B-cell non-Hodgkin’s lymphoma (NHL) can result in disease remission (Zinzani 2003; Czuczman et al. 2004), these therapeutic strategies do not prevent eventual relapse of NHL in most patients (Laudi et al. 2005). New treatment options for lymphoma are desperately needed (Kostelny et al. 2001; Takacs et al. 2004). Rituximab, an anti-CD20 chimeric murine/human monoclonal antibody (mAb), was approved by the US Food and Drugs Administration as the first therapeutic monoclonal antibody against NHL (Pescovitz 2006). Despite the unprecedented success in rituximab treatment of NHL patients, rituximab as a single agent or in combination with cytotoxic chemotherapy agents can still not care the majority of B-cell NHL patients and relapsed patients (Cheson and Leonard 2008). Therefore, development of new therapeutic reagents is urgently needed for improving clinical efficacy. HLA-DR antigen is an attractive target for antibody-based lymphoma therapy because it is highly expressed on a range of hematologic malignancies, and it is neither shed nor internalized after antibody binding (Liu et al. 2004). Antibody can induce antibody-dependent cellular cytotoxicity (ADCC)
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or complement-dependent cytotoxicity (CDC), dependent on competent immune effector cells and functional complement, respectively (Hertlein and Byrd 2010). However, antiHLA-DR mAbs can directly induce cytotoxicity in addition to mediating ADCC and CDC (Coral et al. 1997; Mourad et al. 1990). HL243γ1 is a humanized IgG1 anti-HLA-DR mAb, recognizing a conformational epitope in the α-chain of HLA-DR. Unlike type I rituximab-like anti-CD20 mAbs, hL243γ1 can kill neoplastic cells more potently, independent of CDC (Stein et al. 2006). CD20 and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas. Furthermore, CD20 and HLA-DR are physically and functionally coupled on human B-cell lines (Leveille et al. 1999, 2002) and also share common intracellular signaling pathways (Dechant et al. 2003; Hofmeister et al. 2000; Drenou et al. 1999). However, HLA-DR is also expressed on non-tumor antigen-presenting cells, such as B cells, dendritic cells, and macrophages, which raises the safety concerns of antiHLA-DR antibody for clinical therapies (Stein et al. 2006; Dechant et al. 2003; Broxmeyer et al. 1984). Bispecific antibodies (BsAbs) simultaneously bind to two different antigens and may have the potential to improve the efficacy of cancer immunotherapy (Staerz et al. 1985). BsAbs usually do not occur in nature, but can be generated by genetic engineering (van Spriel et al. 2000; Xie et al. 2005). There are different types of BsAbs, including bispecific single-chain Fv antibody derivatives (bsscFvs) (Kuo et al. 2012; Loffler et al. 2000), tandem single-chain triplebodies (sctbs) (Singer et al. 2010), CrossMabs (Kienast et al. 2013), DVD-Igs (DiGiammarino et al. 2012; Wu et al. 2009), and others. Accordingly, we hypothesize that a bispecific antibody against both HLA-DR and CD20 may have a potent and selective cytotoxicity against NHL. In this study, we developed an anti-CD20/HLA-DR BsAb, CD20-HLA-DR DVD-Ig, derived from rituximab and hL243γ1 by conventional techniques. The CD20-HLADR DVD-Ig simultaneously bound to CD20 and HLADR on B-cell lymphoma induced strong ADCC and CDC against B-cell lymphoma cells by eliciting actin reorganization and homotypic adhesion. More importantly, CD20HLA-DR DVD-Ig exhibited not only a great cytotoxicity, but also high selectivity against CD20+HLA-DR+ NHL cells, suggesting that it may be a promising therapeutic agent for treatment of B-cell lymphoma.
J Cancer Res Clin Oncol
cultured at 37 °C in 5 % CO2 in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. FreeStyle™293-F cells were obtained from Life Technologies and cultured in FreeStyle 293 Expression Medium. Construction of the bispecific antibody The gene for bispecific antibody CD20-HLA-DR DVDIg was constructed using the method described previously (Wu et al. 2007, 2009). Briefly, the DNA fragments for the heavy chain variable region (VH) and light chain variable region (VL) of rituximab were genetically fused to the 5′-terminus of the genes for the heavy chain and light chain of hL243γ1 through a long linker, respectively. The linkers between the two variable regions in the heavy chain (the linker sequence for ASTKGPSVFPLAP) and the light chain (the linker sequence for TVAAPSVFIFPP) were selected from the N-termini of human CH1 and Cκ sequences, respectively. Expression, purification, and verification of the bispecific antibody The cDNAs for the light chain and heavy chain of DVD-Ig were cloned separately into expression vectors by a homologous recombination technology using CloneEZ PCR Cloning Kit, according to the manufacturers’ instruction (GenScript Corporation). The BsAb was produced by transient expression of these heavy and light chain-expressing vectors in FreeStyle™293-F cells, according to the FreeStyle™ 293-F Cells User Manual using Endofree Plasmid Maxi (Qiagen) preparations of the antibody vectors, OptiMEM I medium (life Technologies), PEI (Polysciences), and an initial cell density of 1.2–1.5 × 106 viable cells/ ml in FreeStyle 293 Expression Medium. Seven days later, when cell viability dropped to 50–60 %, the supernatants of cultured cells were harvested. The DVD-Ig antibody in the harvested supernatants was purified by Protein A affinity chromatography (GE Healthcare). The concentrations of DVD-Ig antibody were determined by measuring their absorbance at 280 nm. The purified antibody was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12 % gels under a reducing condition and on 8 % SDS-PAGE under a non-reducing condition, and stained by Coomassie Brilliant Blue.
Materials and methods Cell binding assays Cell culture Burkitt human lymphoma lines (Raji and Ramos) were purchased from American Type Culture Collection and
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The binding of CD20-HLA-DR DVD-Ig to Raji and Ramos cells was determined by flow cytometry on a FACScan (Beckman Coulter, America) using the parental
J Cancer Res Clin Oncol
antibodies of rituximab and hL243γ1 as the positive controls while anti-HER2 mAb as the negative control. Briefly, Raji and Ramos cells (1 × 104/well) were incubated in duplicate with different concentrations of antibodies tested in 100 μl BSA-PBS at 4 °C for 45 min. After centrifugation and being washed, the cells were stained with Alexa Fluor® 488 goat antihuman IgG (H + L) (Life Technologies) 1:1000 in 1 % BSA-PBS at 4 °C for 30 min in the dark. After being washed, the cells were analyzed by flow cytometry. Cytotoxicity assays
Cell death assay Raji and Ramos cells (1 × 105 cells/well) were treated with 10 μg/ml of rituximab, 11B8, hL243γ1, CD20-HLA-DR DVD-Ig, or rituximab + hL243γ1 (rituximab + hL243γ1#, a mixture of rituximab and hL243γ1, 1:1 were 10 μg/ml and rituximab + hL243γ1*, a mixture of rituximab and hL243γ1, 1:1 were 20 μg/ml) in 48-well plates at 37 °C for 48 h. Anti-HER2 mAb was used as a negative control. After being washed, the cells were stained with SYTOX® Red dead cell staining reagent (Life Technologies), washed, and analyzed by flow cytometry using a Flow Cytometer Cytomics FC 500 (Beckman Coulter).
The ability of CD20-HLA-DR DVD-Ig to induce CDC or ADCC was tested. Briefly, Raji and Ramos cells (5 × 104 cells/well) were incubated in triplicate with different concentrations of antibodies (anti-HER2 mAb as a negative control) in phenol red-free DMEM culture medium in 48-well plates in a 5 % CO2 incubator at 37 °C for 1 h. The cells were treated with either normal human serum (NHS, 10 % vol/vol) as a source of complement for CDC assay or human peripheral blood mononuclear cells (PBMCs) as effector cells for ADCC assay at 37 °C for 4 h. The cells were treated with NHS or PBMCs and served as the negative controls. Subsequently, individual wells were added with 50 µl of the CytoTox-Glo™ Cytotoxicity Assay Reagent (Promega). After a brief mix, the plate was incubated at room temperature for 15 min and the dead cell signals were measured using a Berthold LB 960 Centro Microplate Luminometer.
Blood samples from healthy volunteers were obtained under a protocol approved by the Institutional Review Board of Chinese PLA General Hospital. Raji cells (5 × 104/tube) were mixed with heparinized whole blood (150 μl) and incubated in triplicate with the antibodies tested or anti-HER2 mAb (a negative control) at 1 nmol/l at 37 °C and 5 % CO2 for 2 days. The red blood cells were lyzed, and after being washed, the remaining cells were stained with FITC-antiCD19, PC5-anti-CD14, PC7-anti-CD3 (Beckman Coulter, Brea, CA), or APC-conjugated mouse IgG1 isotype control (BD Biosciences) and analyzed by flow cytometry.
Assessment of homotypic adhesion
Antibody generation and characterization
Ramos cells (1 × 105 cells/well) were treated in triplicate with 10 μg/ml of antibodies tested in 300 μl medium in 24-well plates at 37 °C for 4 h. Anti-HER2 mAb was used as a negative control. Homotypic adhesion (HA) of the cells was imaged using an Olympus CKX41 inverted microscope.
We engineered the bispecific CD20-HLA-DR DVD-Ig using the DAN fragments for rituximab and hL243γ1 (Fig. 1A). Under a non-reducing condition, the intact DVD-Ig protein displayed a band with molecular weight of 200~250 kDa (Fig. 1B, lane 3, 4), while a monospecific IgG1 antibody showed ~170 kDa. Under a reducing condition, the bispecific antibody separated into two bands with molecular masses of ~64 kDa (heavy chain) and ~36 kDa (light chain), while a monospecific IgG1 antibody showed a ~50 kDa (heavy chain) and a 25 kDa (light chain).
Assessment of the actin cytoskeleton Ramos cells were treated in triplicate with 10 μg/ml of antibodies tested for 4 h (anti-HER2 mAb was used as a negative control), and cytospun on glass slides that had been coated with poly-l-lysine, followed by fixing them with fresh 2 % paraformaldehyde in PBS for 15 min. The cells were permeabilized with 0.1 % Triton X-100 for 5 min and stained with Alexa Flour 546 phalloidin (Molecular Probes) overnight at 4 °C to label the actin filaments. After being washed, the slides were mounted with DAPI (Life Technology) and examined under an Olympus laser scanning confocal microscope.
Ex vivo depletion of Raji cells from whole blood
Results
Cell binding The binding affinities of CD20-HLA-DR DVD-Ig, rituximab, 11B8, and hL243γ1 to Raji and Ramos cells were measured. The binding affinities of CD20-HLA-DR DVDIg to Ramos were indistinguishable from that of the parental mAb of hL243γ1 and slightly higher than that of the rituximab (Fig. 2A). Furthermore, the binding affinities of CD20-HLA-DR DVD-Ig to Raji cells were similar to that
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Fig. 1 Generation and characterization of bispecific antibodies. A Schematic illustration of the structures of parental rituximab, hL243γ1, and bispecific CD20-HLA-DR DVD-Ig. B SDS-PAGE analysis of the purified antibodies under reducing and non-reducing conditions. Lane M protein markers; lane 1 rituximab (2 μg); lane 2 rituximab (1 μg); lane 3 CD20HLA-DR DVD-Ig (2 μg); lane 4 CD20-HLA-DR DVD-Ig (1 μg); lane 5 hL243γ1 (2 μg); lane 6 hL243γ1 (1 μg). Data are representative images from three separate experiments
Fig. 2 Binding activity of bispecific antibodies to NHL cells. Ramos (A) and Raji (B) cells were treated in triplicate with the indicated concentrations of antibodies at 4 °C for 45 min. Results are shown as mean ± SD of three independent experiments
of the hL243γ1 and were obviously higher than that of the rituximab and 11B8 (Fig. 2B). The levels of HLA-DR expression are ~sixfold higher than that of CD20 on Raji cells, allowing more binding of CD20-HLA-DR DVD-Ig and hL243γ1 than rituximab and 11B8 (Stein et al. 2010). However, Ramos cells express a relatively lower level of HLA-DR (Stein et al. 2006), which may explain a less binding of both parental mAbs and DVD-Ig than that on Raji. These data indicated that CD20-HLA-DR DVD-Ig retained the antigen-binding activity of both parental mAbs.
different concentrations of CD20-HLA-DR DVD-Ig and the same dose of NHS triggered Raji cell death in dose-dependent manner and its CDC activities were similar to that of rituximab and hL243γ1. Similarly, treatment with different doses of CD20-HLA-DR DVD-Ig and the same numbers of PBMCs also induced Raji cell death in a dose-dependent manner and its ADCC activities were indistinguishable from that of rituximab and hL243γ1 (Fig. 3B). These data indicated that CD20-HLA-DR DVD-Ig had strong ADCC and CDC against CD20+HLA-DR+ lymphoma cells.
CD20‑HLA‑DR DVD‑Ig displays potent CDC and ADCC against B‑cell lymphoma
CD20‑HLA‑DR DVD‑Ig induces actin reorganization and homotypic adhesion on B‑cell lymphoma cells
The cytotoxicity of CD20-HLA-DR DVD-Ig against Raji cells was assessed. As shown in Fig. 3A, treatment with
Earlier observations have demonstrated that type II antiCD20 mAbs and anti-HLA-DR mAbs can induce actin
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Fig. 3 CDC and ADCC activities of CD20-HLA-DR DVD-Ig against lymphoma cells. Raji cells were treated in triplicate with the indicated concentrations of antibodies in the presence of normal human serum (complement) or the same numbers of PBMCs (at E:T ratio of 25:1) at 37 °C for 4 h and stained with CytoTox-Glo. The CDC
and ADCC activities of individual types of antibodies against Raji cells were analyzed by flow cytometry. Data are expressed as the mean ± SD of each group of cells from three separate experiments. A The CDC activity. B The ADCC activity
reorganization and HA, associated with cell death (Honeychurch et al. 2012; Ivanov et al. 2009). To understand the potential mechanisms underlying the action of CD20HLA-DR DVD-Ig, the impact of CD20-HLA-DR DVD-Ig on actin reorganization and HA in Ramos cells was determined. We did not test it on Raji cells because Raji cells usually form some loose clusters in a condition that affects the assessment of HA and actin cytoskeleton (BraeschAndersen et al. 1989). Ramos cells were treated with different antibodies and stained with SYTOX red for dead cells. Treatment with type II anti-CD20 antibody (11B8), hL243γ1, or CD20-HLA-DR DVD-Ig, but not type I antiCD20 mAb (rituximab), evoked HA (Fig. 4A) and triggered higher percentages of cell death (Fig. 4C, D). Similarly, treatment with 11B8, hL243γ1, or CD20-HLA-DR DVD-Ig induced obvious actin reorganization in Ramos cells (Fig. 4B). Therefore, CD20-HLA-DR DVD-Ig induced actin reorganization and HA in Ramos cells.
Discussion
Ex vivo depletion of NHL from whole human blood As shown in Fig. 5, treatment with CD20-HLA-DR DVDIg depleted 85 % of Raji cells in whole blood (ex vivo), which was more effective than with rituximab (46 %), 11B8 (69 %), or hL243γ1 (56 %). Furthermore, the percentage of B cells in the CD20-HLA-DR DVD-Ig-treated samples was 72 %, which was slightly higher than 45 % in the rituximab, 43 % in the 11B8, and 44 % in the hL243γ1treated samples. There is no significant difference in the frequency of T cells or monocytes among different groups of cells. Apparently, CD20-HLA-DR DVD-Ig had potent cytotoxicity against Raji cells and less toxicity against normal B cells, as compared with that of other tested antibodies in our experimental condition.
Humanized monoclonal antibodies have been used for successful treatment of patients with hematologic diseases and solid tumors (Adams and Weiner 2005; Weiner et al. 2009). However, some patients do not respond, and relapse of these diseases remains a serious challenge. Hence, development of new therapeutic reagents is urgently needed (Sliwkowski and Mellman 2013; Scott et al. 2012; Carter 2001). The bispecific antibody with dual targeting may preferentially target cancer cells (Schubert and Fey 2012). Currently, there are more than 45 formats of available recombinant bispecific antibodies (Muller and Kontermann 2010), and many of them have the potential to be a promising therapeutic agent for treatment of lymphoma and/or other hematologic malignancies. Blinatumomab, a CD19/ CD3-bispecific T-cell receptor-engaging (BiTE) antibody, has potent cytotoxicity against lymphoma and is a promising candidate for a clinical trial in patients with NHL (Loffler et al. 2000; Hoffman and Gore 2014; Portell et al. 2013; Advani 2011). C2-2b-2b, a bispecific antibody IFNα2b immunocytokine targeting CD20 and HLA-DR, has been used for therapy of many hematopoietic neoplasias, including lymphoma, leukemia, and myeloma (Rossi et al. 2010, 2011). Anti-CD30/anti-CD16 diabody can activate NK cells to lyze CD30+ Hodgkin’s lymphoma and result in a remission of Hodgkin’s disease refractory to chemo- and radiotherapy (Arndt et al. 1999). In the present study, we successfully constructed and characterized a format of genetically engineered BsAb CD20-HLA-DR DVD-Ig for targeting CD20 and HLA-DR (Wu et al. 2007). Firstly, the dual-variable-domain immunoglobulin DVD-Ig protein was a dual-specific, tetravalent IgG-like molecule, which was engineered from the
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Fig. 4 CD20-HLA-DR DVD-Ig elicits actin reorganization and homotypic adhesion and triggers cell death in B-cell lymphoma. In A, B, a negative control, b rituximab, c11B8, d hL243γ1, e CD20-HLADR DVD-Ig. A The extent of HA induced by antibodies (10 μg/ml) was assessed by low-magnification light microscopy 4 h after treatment, and HA in Ramos cells is shown as an example. B Cells were stained for actin using Alexa Flour 546 phalloidin and examined by a confocal microscopy. White arrowheads in c, d and e show actins
that cross the bridge between aggregating cells. C, D Cell death in Ramos and Raji cells, respectively. Cells were treated for 48 h before quantification of the percentage of SYTOX® Red-positive cells by flow cytometry. Anti-HER2 mAb, rituximab, 11B8, hL243γ1, and CD20-HLA-DR DVD-Ig were 10 μg/ml; rituximab + hL243γ1 (rituximab + hL243γ1#, a mixture of rituximab and hL243γ1, 1:1 were 10 μg/ml and rituximab + hL243γ1*, a mixture of rituximab and hL243γ1, 1:1 were 20 μg/ml) were 20 μg/ml as indicated in C, D
DNA fragments for two mAbs with distinct specificity (Wu et al. 2007, 2009). Secondly, the DVD-Ig protein was efficiently produced by conventional mammalian expression systems as a single species for easy manufacture and purification, while it maintains the affinities and potencies of its two parental mAbs (Kou et al. 2010; Tan et al. 2013). Finally, it was highly stable and exhibited excellent IgGlike physicochemical and pharmacokinetic properties (Wu et al. 2007). We found that the bispecific CD20-HLA-DR DVD-Ig bound to CD20 and HLA-DR on B-cell lymphoma with an affinity similar to that of its parental antibodies of rituximab and hL243γ1. The CD20-HLA-DR DVD-Ig
induced potent CDC and ADCC against B-cell lymphoma and elicited HA and actin reorganization in B-cell lymphoma. More importantly, CD20-HLA-DR DVD-Ig preferably depleted CD20+HLA-DR+ Raji lymphoma cells from whole blood, but had a little toxicity against normal B cells although HLA-DR is expressed on normal antigenpresenting cells (Stein et al. 2011). These observations suggest that CD20-HLA-DR DVD-Ig may have highly specific anti-tumor activities. Previous studies have demonstrated that this strategy can overcome the limitations of rituximab (CD20) and cetuximab (epidermal growth factor receptor) as well as many other Abs under clinical
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oxidase, leading to non-apoptotic cell death (Stein et al. 2010; Honeychurch et al. 2012; Alduaij et al. 2011). It is possible that CD20-HLA-DR DVD-Ig may trigger lymphoma cell death by the similar mechanisms or by inducing necroptosis. In conclusion, our data indicated that the genetically engineered BsAb CD20-HLA-DR DVD-Ig bound to both CD20 and HLA-DR and displayed preferably cytotoxicity against B-cell lymphoma cells. The developed new reagent may aid in design of immunotherapies for B-cell lymphoma. Conflict of interest None.
References Fig. 5 Depletion of NHL cells from whole blood by antibodies. Fresh heparinized human blood was mixed with Raji and incubated in triplicate with 1 nmol/l of the indicated mAbs or CD20-HLA-DR DVD-Ig for 2 days. The remaining cells were stained with fluorescent antibodies, and the percentages of remaining Raji, B, T cell, and monocytes were determined by flow cytometry. Data are expressed as the mean ± SD of individual groups of cells from three separate experiments
investigation because the antigens recognized by these antibodies are widely expressed on normal cells (Advani 2011; Tan et al. 2013; Stein et al. 2011). Although CD20 expression is largely limited to B-cell lymphoma, HLA-DR is highly expressed on a variety of hematologic malignancies (Stein et al. 2010; Verfaillie et al. 1992; Demidem et al. 1997). The bispecific CD20-HLA-DR DVD-Ig specifically targeted both CD20 and HLA-DR and may be valuable for immunotherapies for other hematopoietic malignancies. Previous study has revealed that anti-HLA-DR mAbs can efficiently induce cell death directly by modulating the relevant cell signaling and can enhance the potency of rituximab (Stein et al. 2006). Emerging evidence indicates that ligating CD20 and HLA-DR on the surface of malignant B cells with mAbs induces strong HA and subsequent cell death (Honeychurch et al. 2012; Ivanov et al. 2009; Alduaij et al. 2011). Specifically, only type II, but not type I, anti-CD20 mAbs evoke strong HA (and cell death), and anti-HLA-DR has a similar function (Ivanov et al. 2009). The mAb-mediated Fc-dependent tumor clearance has been extensively studied (Uchida et al. 2004; Glennie et al. 2007). We found that the CD20-HLA-DR DVD-Ig effectively triggered lymphoma cell death. It has been demonstrated that mAb ligation, including type II anti-CD20 mAbs (tositumomab and GA101) and anti-HLA-DR mAbs (L243 and 1D10/apolizumab), results in HA and actin reorganization, which can permeabilize the lysosomal membrane, release cathepsins, and generate ROS by NAPDH
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ORIGINAL ARTICLE – CANCER RESEARCH
A bispecific antibody directly induces lymphoma cell death by simultaneously targeting CD20 and HLA‑DR Jing Zeng · Ran Liu · Jinjing Wang · Yi Fang
Received: 13 July 2014 / Accepted: 23 February 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose Both CD20 and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas and are therapeutic targets in antibody-based lymphoma therapy. The aim of this study was to evaluate the anti-tumor effect of a bispecific antibody CD20-HLA-DR DVD-Ig on B-cell lymphoma. Methods The gene for bispecific antibody CD20HLA-DR DVD-Ig was constructed and expressed in FreeStyle™293-F cells, followed by purification. Their functions were characterized for binding to CD20 and HLA-DR and for cytotoxicity against B-cell lymphoma. Results The bispecific antibody CD20-HLA-DR DVDIg was engineered using the DNA fragments for the antiCD20 rituximab and anti-HLA-DR hL243γ1. The CD20HLA-DR DVD-Ig bound simultaneously to both CD20 and HLA-DR, induced potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against B-cell lymphoma, and elicited homotypic adhesion and actin reorganization. Treatment of a mixture of human whole blood and Raji cells with CD20-HLA-DR
Jing Zeng contributed firstly to this work. J. Zeng · R. Liu · J. Wang · Y. Fang (*) Endocrinology Department, Affiliated Hospital of the Academy of Military Medical Sciences, 8 Dongdajie, Beijing 100071, People’s Republic of China e-mail: [email protected] J. Zeng e-mail: [email protected] J. Zeng Cancer Center, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China
DVD-Ig effectively depleted Raji cells and had a little toxicity against normal B cells. Conclusion Our data indicated that targeting both CD20 and HLA-DR was an effective way against NHL, suggesting that CD20-HLA-DR DVD-Ig may be a promising therapeutic agent for B-cell lymphoma. Keywords Bispecific antibody · Cell death · CD20 · HLA-DR · Lymphoma
Introduction Although traditional approaches for treatment of B-cell non-Hodgkin’s lymphoma (NHL) can result in disease remission (Zinzani 2003; Czuczman et al. 2004), these therapeutic strategies do not prevent eventual relapse of NHL in most patients (Laudi et al. 2005). New treatment options for lymphoma are desperately needed (Kostelny et al. 2001; Takacs et al. 2004). Rituximab, an anti-CD20 chimeric murine/human monoclonal antibody (mAb), was approved by the US Food and Drugs Administration as the first therapeutic monoclonal antibody against NHL (Pescovitz 2006). Despite the unprecedented success in rituximab treatment of NHL patients, rituximab as a single agent or in combination with cytotoxic chemotherapy agents can still not care the majority of B-cell NHL patients and relapsed patients (Cheson and Leonard 2008). Therefore, development of new therapeutic reagents is urgently needed for improving clinical efficacy. HLA-DR antigen is an attractive target for antibody-based lymphoma therapy because it is highly expressed on a range of hematologic malignancies, and it is neither shed nor internalized after antibody binding (Liu et al. 2004). Antibody can induce antibody-dependent cellular cytotoxicity (ADCC)
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or complement-dependent cytotoxicity (CDC), dependent on competent immune effector cells and functional complement, respectively (Hertlein and Byrd 2010). However, antiHLA-DR mAbs can directly induce cytotoxicity in addition to mediating ADCC and CDC (Coral et al. 1997; Mourad et al. 1990). HL243γ1 is a humanized IgG1 anti-HLA-DR mAb, recognizing a conformational epitope in the α-chain of HLA-DR. Unlike type I rituximab-like anti-CD20 mAbs, hL243γ1 can kill neoplastic cells more potently, independent of CDC (Stein et al. 2006). CD20 and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas. Furthermore, CD20 and HLA-DR are physically and functionally coupled on human B-cell lines (Leveille et al. 1999, 2002) and also share common intracellular signaling pathways (Dechant et al. 2003; Hofmeister et al. 2000; Drenou et al. 1999). However, HLA-DR is also expressed on non-tumor antigen-presenting cells, such as B cells, dendritic cells, and macrophages, which raises the safety concerns of antiHLA-DR antibody for clinical therapies (Stein et al. 2006; Dechant et al. 2003; Broxmeyer et al. 1984). Bispecific antibodies (BsAbs) simultaneously bind to two different antigens and may have the potential to improve the efficacy of cancer immunotherapy (Staerz et al. 1985). BsAbs usually do not occur in nature, but can be generated by genetic engineering (van Spriel et al. 2000; Xie et al. 2005). There are different types of BsAbs, including bispecific single-chain Fv antibody derivatives (bsscFvs) (Kuo et al. 2012; Loffler et al. 2000), tandem single-chain triplebodies (sctbs) (Singer et al. 2010), CrossMabs (Kienast et al. 2013), DVD-Igs (DiGiammarino et al. 2012; Wu et al. 2009), and others. Accordingly, we hypothesize that a bispecific antibody against both HLA-DR and CD20 may have a potent and selective cytotoxicity against NHL. In this study, we developed an anti-CD20/HLA-DR BsAb, CD20-HLA-DR DVD-Ig, derived from rituximab and hL243γ1 by conventional techniques. The CD20-HLADR DVD-Ig simultaneously bound to CD20 and HLADR on B-cell lymphoma induced strong ADCC and CDC against B-cell lymphoma cells by eliciting actin reorganization and homotypic adhesion. More importantly, CD20HLA-DR DVD-Ig exhibited not only a great cytotoxicity, but also high selectivity against CD20+HLA-DR+ NHL cells, suggesting that it may be a promising therapeutic agent for treatment of B-cell lymphoma.
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cultured at 37 °C in 5 % CO2 in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. FreeStyle™293-F cells were obtained from Life Technologies and cultured in FreeStyle 293 Expression Medium. Construction of the bispecific antibody The gene for bispecific antibody CD20-HLA-DR DVDIg was constructed using the method described previously (Wu et al. 2007, 2009). Briefly, the DNA fragments for the heavy chain variable region (VH) and light chain variable region (VL) of rituximab were genetically fused to the 5′-terminus of the genes for the heavy chain and light chain of hL243γ1 through a long linker, respectively. The linkers between the two variable regions in the heavy chain (the linker sequence for ASTKGPSVFPLAP) and the light chain (the linker sequence for TVAAPSVFIFPP) were selected from the N-termini of human CH1 and Cκ sequences, respectively. Expression, purification, and verification of the bispecific antibody The cDNAs for the light chain and heavy chain of DVD-Ig were cloned separately into expression vectors by a homologous recombination technology using CloneEZ PCR Cloning Kit, according to the manufacturers’ instruction (GenScript Corporation). The BsAb was produced by transient expression of these heavy and light chain-expressing vectors in FreeStyle™293-F cells, according to the FreeStyle™ 293-F Cells User Manual using Endofree Plasmid Maxi (Qiagen) preparations of the antibody vectors, OptiMEM I medium (life Technologies), PEI (Polysciences), and an initial cell density of 1.2–1.5 × 106 viable cells/ ml in FreeStyle 293 Expression Medium. Seven days later, when cell viability dropped to 50–60 %, the supernatants of cultured cells were harvested. The DVD-Ig antibody in the harvested supernatants was purified by Protein A affinity chromatography (GE Healthcare). The concentrations of DVD-Ig antibody were determined by measuring their absorbance at 280 nm. The purified antibody was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12 % gels under a reducing condition and on 8 % SDS-PAGE under a non-reducing condition, and stained by Coomassie Brilliant Blue.
Materials and methods Cell binding assays Cell culture Burkitt human lymphoma lines (Raji and Ramos) were purchased from American Type Culture Collection and
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The binding of CD20-HLA-DR DVD-Ig to Raji and Ramos cells was determined by flow cytometry on a FACScan (Beckman Coulter, America) using the parental
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antibodies of rituximab and hL243γ1 as the positive controls while anti-HER2 mAb as the negative control. Briefly, Raji and Ramos cells (1 × 104/well) were incubated in duplicate with different concentrations of antibodies tested in 100 μl BSA-PBS at 4 °C for 45 min. After centrifugation and being washed, the cells were stained with Alexa Fluor® 488 goat antihuman IgG (H + L) (Life Technologies) 1:1000 in 1 % BSA-PBS at 4 °C for 30 min in the dark. After being washed, the cells were analyzed by flow cytometry. Cytotoxicity assays
Cell death assay Raji and Ramos cells (1 × 105 cells/well) were treated with 10 μg/ml of rituximab, 11B8, hL243γ1, CD20-HLA-DR DVD-Ig, or rituximab + hL243γ1 (rituximab + hL243γ1#, a mixture of rituximab and hL243γ1, 1:1 were 10 μg/ml and rituximab + hL243γ1*, a mixture of rituximab and hL243γ1, 1:1 were 20 μg/ml) in 48-well plates at 37 °C for 48 h. Anti-HER2 mAb was used as a negative control. After being washed, the cells were stained with SYTOX® Red dead cell staining reagent (Life Technologies), washed, and analyzed by flow cytometry using a Flow Cytometer Cytomics FC 500 (Beckman Coulter).
The ability of CD20-HLA-DR DVD-Ig to induce CDC or ADCC was tested. Briefly, Raji and Ramos cells (5 × 104 cells/well) were incubated in triplicate with different concentrations of antibodies (anti-HER2 mAb as a negative control) in phenol red-free DMEM culture medium in 48-well plates in a 5 % CO2 incubator at 37 °C for 1 h. The cells were treated with either normal human serum (NHS, 10 % vol/vol) as a source of complement for CDC assay or human peripheral blood mononuclear cells (PBMCs) as effector cells for ADCC assay at 37 °C for 4 h. The cells were treated with NHS or PBMCs and served as the negative controls. Subsequently, individual wells were added with 50 µl of the CytoTox-Glo™ Cytotoxicity Assay Reagent (Promega). After a brief mix, the plate was incubated at room temperature for 15 min and the dead cell signals were measured using a Berthold LB 960 Centro Microplate Luminometer.
Blood samples from healthy volunteers were obtained under a protocol approved by the Institutional Review Board of Chinese PLA General Hospital. Raji cells (5 × 104/tube) were mixed with heparinized whole blood (150 μl) and incubated in triplicate with the antibodies tested or anti-HER2 mAb (a negative control) at 1 nmol/l at 37 °C and 5 % CO2 for 2 days. The red blood cells were lyzed, and after being washed, the remaining cells were stained with FITC-antiCD19, PC5-anti-CD14, PC7-anti-CD3 (Beckman Coulter, Brea, CA), or APC-conjugated mouse IgG1 isotype control (BD Biosciences) and analyzed by flow cytometry.
Assessment of homotypic adhesion
Antibody generation and characterization
Ramos cells (1 × 105 cells/well) were treated in triplicate with 10 μg/ml of antibodies tested in 300 μl medium in 24-well plates at 37 °C for 4 h. Anti-HER2 mAb was used as a negative control. Homotypic adhesion (HA) of the cells was imaged using an Olympus CKX41 inverted microscope.
We engineered the bispecific CD20-HLA-DR DVD-Ig using the DAN fragments for rituximab and hL243γ1 (Fig. 1A). Under a non-reducing condition, the intact DVD-Ig protein displayed a band with molecular weight of 200~250 kDa (Fig. 1B, lane 3, 4), while a monospecific IgG1 antibody showed ~170 kDa. Under a reducing condition, the bispecific antibody separated into two bands with molecular masses of ~64 kDa (heavy chain) and ~36 kDa (light chain), while a monospecific IgG1 antibody showed a ~50 kDa (heavy chain) and a 25 kDa (light chain).
Assessment of the actin cytoskeleton Ramos cells were treated in triplicate with 10 μg/ml of antibodies tested for 4 h (anti-HER2 mAb was used as a negative control), and cytospun on glass slides that had been coated with poly-l-lysine, followed by fixing them with fresh 2 % paraformaldehyde in PBS for 15 min. The cells were permeabilized with 0.1 % Triton X-100 for 5 min and stained with Alexa Flour 546 phalloidin (Molecular Probes) overnight at 4 °C to label the actin filaments. After being washed, the slides were mounted with DAPI (Life Technology) and examined under an Olympus laser scanning confocal microscope.
Ex vivo depletion of Raji cells from whole blood
Results
Cell binding The binding affinities of CD20-HLA-DR DVD-Ig, rituximab, 11B8, and hL243γ1 to Raji and Ramos cells were measured. The binding affinities of CD20-HLA-DR DVDIg to Ramos were indistinguishable from that of the parental mAb of hL243γ1 and slightly higher than that of the rituximab (Fig. 2A). Furthermore, the binding affinities of CD20-HLA-DR DVD-Ig to Raji cells were similar to that
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Fig. 1 Generation and characterization of bispecific antibodies. A Schematic illustration of the structures of parental rituximab, hL243γ1, and bispecific CD20-HLA-DR DVD-Ig. B SDS-PAGE analysis of the purified antibodies under reducing and non-reducing conditions. Lane M protein markers; lane 1 rituximab (2 μg); lane 2 rituximab (1 μg); lane 3 CD20HLA-DR DVD-Ig (2 μg); lane 4 CD20-HLA-DR DVD-Ig (1 μg); lane 5 hL243γ1 (2 μg); lane 6 hL243γ1 (1 μg). Data are representative images from three separate experiments
Fig. 2 Binding activity of bispecific antibodies to NHL cells. Ramos (A) and Raji (B) cells were treated in triplicate with the indicated concentrations of antibodies at 4 °C for 45 min. Results are shown as mean ± SD of three independent experiments
of the hL243γ1 and were obviously higher than that of the rituximab and 11B8 (Fig. 2B). The levels of HLA-DR expression are ~sixfold higher than that of CD20 on Raji cells, allowing more binding of CD20-HLA-DR DVD-Ig and hL243γ1 than rituximab and 11B8 (Stein et al. 2010). However, Ramos cells express a relatively lower level of HLA-DR (Stein et al. 2006), which may explain a less binding of both parental mAbs and DVD-Ig than that on Raji. These data indicated that CD20-HLA-DR DVD-Ig retained the antigen-binding activity of both parental mAbs.
different concentrations of CD20-HLA-DR DVD-Ig and the same dose of NHS triggered Raji cell death in dose-dependent manner and its CDC activities were similar to that of rituximab and hL243γ1. Similarly, treatment with different doses of CD20-HLA-DR DVD-Ig and the same numbers of PBMCs also induced Raji cell death in a dose-dependent manner and its ADCC activities were indistinguishable from that of rituximab and hL243γ1 (Fig. 3B). These data indicated that CD20-HLA-DR DVD-Ig had strong ADCC and CDC against CD20+HLA-DR+ lymphoma cells.
CD20‑HLA‑DR DVD‑Ig displays potent CDC and ADCC against B‑cell lymphoma
CD20‑HLA‑DR DVD‑Ig induces actin reorganization and homotypic adhesion on B‑cell lymphoma cells
The cytotoxicity of CD20-HLA-DR DVD-Ig against Raji cells was assessed. As shown in Fig. 3A, treatment with
Earlier observations have demonstrated that type II antiCD20 mAbs and anti-HLA-DR mAbs can induce actin
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Fig. 3 CDC and ADCC activities of CD20-HLA-DR DVD-Ig against lymphoma cells. Raji cells were treated in triplicate with the indicated concentrations of antibodies in the presence of normal human serum (complement) or the same numbers of PBMCs (at E:T ratio of 25:1) at 37 °C for 4 h and stained with CytoTox-Glo. The CDC
and ADCC activities of individual types of antibodies against Raji cells were analyzed by flow cytometry. Data are expressed as the mean ± SD of each group of cells from three separate experiments. A The CDC activity. B The ADCC activity
reorganization and HA, associated with cell death (Honeychurch et al. 2012; Ivanov et al. 2009). To understand the potential mechanisms underlying the action of CD20HLA-DR DVD-Ig, the impact of CD20-HLA-DR DVD-Ig on actin reorganization and HA in Ramos cells was determined. We did not test it on Raji cells because Raji cells usually form some loose clusters in a condition that affects the assessment of HA and actin cytoskeleton (BraeschAndersen et al. 1989). Ramos cells were treated with different antibodies and stained with SYTOX red for dead cells. Treatment with type II anti-CD20 antibody (11B8), hL243γ1, or CD20-HLA-DR DVD-Ig, but not type I antiCD20 mAb (rituximab), evoked HA (Fig. 4A) and triggered higher percentages of cell death (Fig. 4C, D). Similarly, treatment with 11B8, hL243γ1, or CD20-HLA-DR DVD-Ig induced obvious actin reorganization in Ramos cells (Fig. 4B). Therefore, CD20-HLA-DR DVD-Ig induced actin reorganization and HA in Ramos cells.
Discussion
Ex vivo depletion of NHL from whole human blood As shown in Fig. 5, treatment with CD20-HLA-DR DVDIg depleted 85 % of Raji cells in whole blood (ex vivo), which was more effective than with rituximab (46 %), 11B8 (69 %), or hL243γ1 (56 %). Furthermore, the percentage of B cells in the CD20-HLA-DR DVD-Ig-treated samples was 72 %, which was slightly higher than 45 % in the rituximab, 43 % in the 11B8, and 44 % in the hL243γ1treated samples. There is no significant difference in the frequency of T cells or monocytes among different groups of cells. Apparently, CD20-HLA-DR DVD-Ig had potent cytotoxicity against Raji cells and less toxicity against normal B cells, as compared with that of other tested antibodies in our experimental condition.
Humanized monoclonal antibodies have been used for successful treatment of patients with hematologic diseases and solid tumors (Adams and Weiner 2005; Weiner et al. 2009). However, some patients do not respond, and relapse of these diseases remains a serious challenge. Hence, development of new therapeutic reagents is urgently needed (Sliwkowski and Mellman 2013; Scott et al. 2012; Carter 2001). The bispecific antibody with dual targeting may preferentially target cancer cells (Schubert and Fey 2012). Currently, there are more than 45 formats of available recombinant bispecific antibodies (Muller and Kontermann 2010), and many of them have the potential to be a promising therapeutic agent for treatment of lymphoma and/or other hematologic malignancies. Blinatumomab, a CD19/ CD3-bispecific T-cell receptor-engaging (BiTE) antibody, has potent cytotoxicity against lymphoma and is a promising candidate for a clinical trial in patients with NHL (Loffler et al. 2000; Hoffman and Gore 2014; Portell et al. 2013; Advani 2011). C2-2b-2b, a bispecific antibody IFNα2b immunocytokine targeting CD20 and HLA-DR, has been used for therapy of many hematopoietic neoplasias, including lymphoma, leukemia, and myeloma (Rossi et al. 2010, 2011). Anti-CD30/anti-CD16 diabody can activate NK cells to lyze CD30+ Hodgkin’s lymphoma and result in a remission of Hodgkin’s disease refractory to chemo- and radiotherapy (Arndt et al. 1999). In the present study, we successfully constructed and characterized a format of genetically engineered BsAb CD20-HLA-DR DVD-Ig for targeting CD20 and HLA-DR (Wu et al. 2007). Firstly, the dual-variable-domain immunoglobulin DVD-Ig protein was a dual-specific, tetravalent IgG-like molecule, which was engineered from the
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Fig. 4 CD20-HLA-DR DVD-Ig elicits actin reorganization and homotypic adhesion and triggers cell death in B-cell lymphoma. In A, B, a negative control, b rituximab, c11B8, d hL243γ1, e CD20-HLADR DVD-Ig. A The extent of HA induced by antibodies (10 μg/ml) was assessed by low-magnification light microscopy 4 h after treatment, and HA in Ramos cells is shown as an example. B Cells were stained for actin using Alexa Flour 546 phalloidin and examined by a confocal microscopy. White arrowheads in c, d and e show actins
that cross the bridge between aggregating cells. C, D Cell death in Ramos and Raji cells, respectively. Cells were treated for 48 h before quantification of the percentage of SYTOX® Red-positive cells by flow cytometry. Anti-HER2 mAb, rituximab, 11B8, hL243γ1, and CD20-HLA-DR DVD-Ig were 10 μg/ml; rituximab + hL243γ1 (rituximab + hL243γ1#, a mixture of rituximab and hL243γ1, 1:1 were 10 μg/ml and rituximab + hL243γ1*, a mixture of rituximab and hL243γ1, 1:1 were 20 μg/ml) were 20 μg/ml as indicated in C, D
DNA fragments for two mAbs with distinct specificity (Wu et al. 2007, 2009). Secondly, the DVD-Ig protein was efficiently produced by conventional mammalian expression systems as a single species for easy manufacture and purification, while it maintains the affinities and potencies of its two parental mAbs (Kou et al. 2010; Tan et al. 2013). Finally, it was highly stable and exhibited excellent IgGlike physicochemical and pharmacokinetic properties (Wu et al. 2007). We found that the bispecific CD20-HLA-DR DVD-Ig bound to CD20 and HLA-DR on B-cell lymphoma with an affinity similar to that of its parental antibodies of rituximab and hL243γ1. The CD20-HLA-DR DVD-Ig
induced potent CDC and ADCC against B-cell lymphoma and elicited HA and actin reorganization in B-cell lymphoma. More importantly, CD20-HLA-DR DVD-Ig preferably depleted CD20+HLA-DR+ Raji lymphoma cells from whole blood, but had a little toxicity against normal B cells although HLA-DR is expressed on normal antigenpresenting cells (Stein et al. 2011). These observations suggest that CD20-HLA-DR DVD-Ig may have highly specific anti-tumor activities. Previous studies have demonstrated that this strategy can overcome the limitations of rituximab (CD20) and cetuximab (epidermal growth factor receptor) as well as many other Abs under clinical
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oxidase, leading to non-apoptotic cell death (Stein et al. 2010; Honeychurch et al. 2012; Alduaij et al. 2011). It is possible that CD20-HLA-DR DVD-Ig may trigger lymphoma cell death by the similar mechanisms or by inducing necroptosis. In conclusion, our data indicated that the genetically engineered BsAb CD20-HLA-DR DVD-Ig bound to both CD20 and HLA-DR and displayed preferably cytotoxicity against B-cell lymphoma cells. The developed new reagent may aid in design of immunotherapies for B-cell lymphoma. Conflict of interest None.
References Fig. 5 Depletion of NHL cells from whole blood by antibodies. Fresh heparinized human blood was mixed with Raji and incubated in triplicate with 1 nmol/l of the indicated mAbs or CD20-HLA-DR DVD-Ig for 2 days. The remaining cells were stained with fluorescent antibodies, and the percentages of remaining Raji, B, T cell, and monocytes were determined by flow cytometry. Data are expressed as the mean ± SD of individual groups of cells from three separate experiments
investigation because the antigens recognized by these antibodies are widely expressed on normal cells (Advani 2011; Tan et al. 2013; Stein et al. 2011). Although CD20 expression is largely limited to B-cell lymphoma, HLA-DR is highly expressed on a variety of hematologic malignancies (Stein et al. 2010; Verfaillie et al. 1992; Demidem et al. 1997). The bispecific CD20-HLA-DR DVD-Ig specifically targeted both CD20 and HLA-DR and may be valuable for immunotherapies for other hematopoietic malignancies. Previous study has revealed that anti-HLA-DR mAbs can efficiently induce cell death directly by modulating the relevant cell signaling and can enhance the potency of rituximab (Stein et al. 2006). Emerging evidence indicates that ligating CD20 and HLA-DR on the surface of malignant B cells with mAbs induces strong HA and subsequent cell death (Honeychurch et al. 2012; Ivanov et al. 2009; Alduaij et al. 2011). Specifically, only type II, but not type I, anti-CD20 mAbs evoke strong HA (and cell death), and anti-HLA-DR has a similar function (Ivanov et al. 2009). The mAb-mediated Fc-dependent tumor clearance has been extensively studied (Uchida et al. 2004; Glennie et al. 2007). We found that the CD20-HLA-DR DVD-Ig effectively triggered lymphoma cell death. It has been demonstrated that mAb ligation, including type II anti-CD20 mAbs (tositumomab and GA101) and anti-HLA-DR mAbs (L243 and 1D10/apolizumab), results in HA and actin reorganization, which can permeabilize the lysosomal membrane, release cathepsins, and generate ROS by NAPDH
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