Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells

Zhang, Libin; Fang, Yixin; Yang, Jiyuan; Kopeček, Jindřich

doi:10.1016/j.jconrel.2016.12.025

Cited by 22 publications

(28 citation statements)

References 67 publications

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“…As the internalization is an indicator for the CD20 crosslinking, this result demonstrated that the multiple binding of P‐(MORF2) 10 to Fab′‐MORF1 that was bound to CD20 facilitated the CD 20 crosslinking, which eventually resulted in elevated level of apoptosis. These data are in agreement with our previous study that demonstrated that the higher the valence of the P‐(MORF2) x conjugate the higher the apoptotic response, due to the enhanced biorecognition.…”

Section: Resultssupporting

confidence: 93%

“…2.5 × 10 5 Raji cells were treated with cell culture medium (control) or consecutive treatment Fab′‐MORF1 (1 × 10 −6 m , 1 h)/P‐(MORF2) x (1 × 10 −6 m , 24 h). Then cells were washed by PBS and stained with Annexin V‐FITC and PI in dark for 15 min, followed by flow cytometry analysis . All experiments were carried out in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…FITC Annexin V (also written as Annexin V-FITC, fluorochrome-labeled Annexin V, Biolegend) and propidium iodide (PI) staining were performed following the RAPID protocol provided by the manufacturer (Oncogene Research Products, Boston, MA washed by PBS and stained with Annexin V-FITC and PI in dark for 15 min, followed by flow cytometry analysis. [10,26] All experiments were carried out in triplicate.…”

Section: Apoptosis Assaymentioning

confidence: 99%

“…

Fc fragments might hold great promise for clinical use.Recently, inspired by the direct initiation of apoptosis after cell-surface receptor clustering, we have developed a twostep pretargeted nanotherapy for CD20 crosslinking (Figure 1). [7][8][9][10][11][12] In this system, the Fc fragment of rituximab was depleted and CD20 crosslinking was achieved based on the biorecognition of (1) a pretargeting component Fab′-MORF1 (anti-CD20 Fab′ conjugated with an morpholino oligonucleotide and (2) a crosslinking component P-(MORF2) X N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide. Results have demonstrated the superior antilymphoma efficacy to rituximab in vivo.

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confidence: 99%

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Drug‐Free Macromolecular Therapeutics Induce Apoptosis via Calcium Influx and Mitochondrial Signaling Pathway

Yang

Wang

et al. 2017

Macromolecular Bioscience

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Recently, an innovative paradigm has been proposed in macromolecular therapeutics for treatment of B-cell lymphomas that can specifically kill cancer cells without a drug. The design rationale of this drug-free macromolecular therapeutic (DFMT) system is crosslinking the cell surface receptor to initiate apoptosis. However, how the apoptosis signal is triggered after receptor hyper-crosslinking remains to be elucidated. Here, two pathways, calcium influx dependent pathway and mitochondrial signal pathway, are identified to play major roles in triggering the programmed cell death. With the first step pretargeting and second step multiple binding, receptor hyper-crosslinking is achieved in a highly specific, time-dependent manner and largely mediated by multivalence. As a consequence, extracellular calcium influx is triggered, which subsequently decreases the mitochondrial membrane potential and induces apoptosis. The mitochondrial depolarization also stems from the Bcl-2 inhibition mediated by DFMT, followed by the cytochrome c release that activates caspase signaling. With the participation of the two-pronged mechanism, a programmed apoptosis is induced in response to DFMT treatment. The current findings can offer important implications to optimize the anti-CD20 strategies to treat B-cell non-Hodgkin lymphomas.

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Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Apoptosis Assaymentioning

confidence: 99%

“…

…”

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confidence: 99%

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Drug‐Free Macromolecular Therapeutics Induce Apoptosis via Calcium Influx and Mitochondrial Signaling Pathway

Yang

Wang

et al. 2017

Macromolecular Bioscience

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“…Previously we have demonstrated the higher the valence, the more efficient and pronounced are CD20 cross-linking and apoptosis induction. 24 We also anticipate once several closely related apoptosis signals are high enough in magnitude, then the intracellular RTX resistance due to abnormal modulation ( e.g ., downregulation of Bax and Bak) 25 might be compensated. Thus, potentially, the two-step CD20 cross-linking amplifiers are engaged in a two-pronged effort to combat RTX resistance: circumventing Fc-related resistance pathways and confronting abnormal modulations with enhanced apoptosis, in a way that is independent of immune effector functions.…”

mentioning

confidence: 99%

Amplification of CD20 Cross-Linking in Rituximab-Resistant B-Lymphoma Cells Enhances Apoptosis Induction by Drug-Free Macromolecular Therapeutics

Yang

Wang

et al. 2018

ACS Nano

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Although the CD20-targeted monoclonal antibody rituximab (RTX) has revolutionized the therapeutic landscape for B-cell malignancy, relapsed and refractory disease due to RTX resistance continue to constitute major challenges, illustrating the need for better therapies. Here, we apply drug-free macromolecular therapeutics (DFMT) that amplifies CD20 cross-linking to enhance apoptosis in RTX-resistant cells. Bispecific engager (anti-CD20 Fab′ conjugated with oligonucleotide1) pretargets CD20 and the deletion of Fc-region minimizes its premature endocytosis in resistant cells that rapidly internalize and consume CD20/RTX complexes. Second-step delivery of multivalent polymeric effector (linear copolymer conjugated with multiple copies of complementary oligonucleotide 2) simultaneously hybridizes multiple CD20-bound engagers and strengthens CD20 ligation. Moreover, the restoration of CD20 expression by the pretreatment of cells with a polymer-gemcitabine conjugate, a CD20 expression enhancer, unleashes the full potential of DFMT in the CD20-deficient resistant cells. Hence, amplification of CD20 cross-linking is achieved by (1) the enhancement of surface CD20 accessibility, (2) the increase in CD20 expression, and (3) multimeric CD20 binding, which ultimately translates into the amplified activation of a wide range of innate apoptotic responses. We demonstrated that the altered molecular signaling pathway that originally results in RTX resistance could be circumvented and compensated by other DFMT-augmented pathways. Of note, our preliminary data provide proof-of-concept that CD20 cross-linking amplification emerges as an important strategy for overcoming RTX resistance.

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Human Serum Albumin‐Based Drug‐Free Macromolecular Therapeutics: Apoptosis Induction by Coiled‐Coil‐Mediated Cross‐Linking of CD20 Antigens on Lymphoma B Cell Surface

Zhang

Fang

et al. 2018

Macromolecular Bioscience

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We have recently developed a therapeutic platform – drug-free macromolecular therapeutics (DFMT) that induces apoptosis in B cells by crosslinking of CD20 receptors, without the need for low molecular weight cytotoxic drug. In this report a DFMT system was synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab’ fragment of anti CD20 monoclonal antibody rituximab (RTX) was attached to CCE (Fab’-CCE); multiple grafts of CCK were conjugated to HSA (HSA-(CCK)7). Confocal fluorescence microscopy demonstrated colocalization of both nanoconjugates at the surface of non-Hodgkin’s lymphoma (NHL) Raji cells. The colocalization led to coiled-coil formation, CD20 crosslinking and apoptosis induction. The apoptotic levels were evaluated by Annexin V, Caspase 3 and TUNEL assays. Selective surface binding of DFMT to CD20+ cells was validated in experiments on a coculture of CD20+ (Raji) and CD20- (DG-75) cells. DMFT could trigger calcium influx only in Raji cells, but not in DG-75 cells. This HSA-based DFMT system presents a highly specific treatment for NHL and other B cell malignancies with considerable translational potential.

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Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells

Cited by 22 publications

References 67 publications

Drug‐Free Macromolecular Therapeutics Induce Apoptosis via Calcium Influx and Mitochondrial Signaling Pathway

Drug‐Free Macromolecular Therapeutics Induce Apoptosis via Calcium Influx and Mitochondrial Signaling Pathway

Amplification of CD20 Cross-Linking in Rituximab-Resistant B-Lymphoma Cells Enhances Apoptosis Induction by Drug-Free Macromolecular Therapeutics

Human Serum Albumin‐Based Drug‐Free Macromolecular Therapeutics: Apoptosis Induction by Coiled‐Coil‐Mediated Cross‐Linking of CD20 Antigens on Lymphoma B Cell Surface

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