Apoptosis of Raji cells by an anti-CD20 antibody HI47 and its fragments

2004 ◽  
Vol 28 (2) ◽  
pp. 209-211 ◽  
Author(s):  
Yin-Xing Liu ◽  
Dong-Sheng Xiong ◽  
Dong-Mei Fan ◽  
Yuan-Fu Xu ◽  
Chun-Zheng Yang
Keyword(s):  
Raji Cells ◽  
Cd20 Antibody ◽  
Anti Cd20

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Maciej Szydlowski ◽  
Filip Garbicz ◽  
Ewa Jabłońska ◽  
Patryk Górniak ◽  
Beata Pyrzynska ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Raji Cells ◽  
Pim Kinases ◽  
Large B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Large B Cell

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

scholarly journals 266 AK117, a CD47 blocking antibody with robust macrophage activation without red blood cell hemagglutination

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A288-A289
Author(s):  
Zhaoliang Huang ◽  
Xinghua Pang ◽  
Tingting Zhong ◽  
Tailong Qu ◽  
Chunshan Jin ◽  
...  
Keyword(s):  
Safety Profile ◽  
Phagocytic Index ◽  
Raji Cells ◽  
Cynomolgus Monkeys ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Anti Tumor Activity ◽  
Ht 29 ◽  
Human Rbcs ◽  
Egfr Antibody

BackgroundAK117 is a humanized monoclonal antibody targeting CD47 which widely expresses on innate immune cells, such as macrophages, and functions as a regulator of phagocytosis. CD47 serves as the ligand for a receptor on these innate immune cells, SIRPα, which in turn delivers an inhibitory signal for phagocytosis. Hematology toxicity is the major concern of an anti-CD47 antibody. As an agent targeting CD47 being investigated as an anti-tumor therapeutic, AK117 is engineered on a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions, as well as identified with favorable hematology safety profile and robust pro-phagocytosis activity.MethodsActivity of AK117 binding to CD47 to block the interaction between CD47 and SIRPα were determined by FACS, and binding of AK117 to human RBC was also evaluated. Raji cells, HT-29 cells, and HL-60 cells which highly express CD47 were used as target cells to evaluate a pro-phagocytic activity of AK117 as a monotherapy or in combination with anti-EGFR antibody, anti-CD20 antibody or azacitidine. In in-vivo pharmacology studies, anti-tumor activity of AK117 was investigated in SCID/beige mouse Raji tumor model. Effects of AK117 on hemagglutination of human RBC at was tested. Hemoglobin (HGB) and hematocrit (HCT) was evaluated after single dose of 10 mg/kg AK117 or Hu-5F9 in male and female cynomolgus monkeys (n=1/gender).ResultsAK117 could effectively binds to CD47, and competes with SIRPα for binding to the antigen on Raji cells (figure 1). AK117 alone or combines with anti-EGFR antibody, anti-CD20 antibody and azacitidine shows potent phagocytosis of tumor cells in a dose-dependent manner (figure 2). AK117 significantly inhibited tumor growth in these tumor models (figure 3). Favorable hematology safety profile of AK117 was observed. A significant weaker binding to human RBC of AK117 was identified (figure 4), and AK117 does not induce hemagglutination of human RBC up to a concentration of 1050 μg/mL, while Hu-5F9 triggers hemagglutination even at a low concentration of 1.44 μg/mL (figure 5). AK117 has minimal anemia effect in monkey studies compared to hu5F9-G4 after single dose in cynomolgus monkeys (figure 6). AK117 showed a rather superior safety profile to Hu5F9-G4 as a shorter duration of anemia.Abstract 266 Figure 1Binding and Competition activity of AK117 to CD47. (A) FACS binding curves of AK117 and Hu5F9-G4 to CD47 on raji cells. (B) FACS competitive binding curve of AK117 and Hu5F9-G4 with SIRPαECD-mFc to CD47 on raji cells.Abstract 266 Figure 2The pro-phagocytic activity against tumor cells. (A) The phagocytic index of raji cells by macrophages with AK117. (B) The phagocytic index of HL-60 cells by macrophages with AK117 and azacitidine. (C) The phagocytic index of HT-29 cells by macrophages with AK117 and cetuximab. (D) The phagocytic index of raji cells by macrophages with AK117 and rituximab.Abstract 266 Figure 3Anti-tumor activity in raji tumor mouse model. The (A) Tumor growth curves and (B) Body weight curves of different groups in SCID/Beige mice with subcutaneous raji tumor.Abstract 266 Figure 4Binding activity of AK117 to human RBCs. Binding Curves of Hu5F9-G4 and AK117 to CD47 on human RBCsAbstract 266 Figure 5Hemagglutination effect on human erythrocytes. Hemagglutination effect of AK117 on human erythrocytesAbstract 266 Figure 6HGB and HCT in cynomolgus monkeys. The curves of (A) hemoglobin and (B) Hematocrit at different times in cynomolgus monkeys.ConclusionsWith pre-clinical pharmacology activities comparable to Hu5F9-G4 as well as superior safety properties demonstrated in non-clinical pharmacodynamics studies, AK117 has emerged as a promising new treatment for solid tumor.

Novel treatment opportunities in Graves’ orbitopathy

2014 ◽  
Vol 155 (33) ◽  
pp. 1295-1300
Author(s):  
Annamária Erdei ◽  
Annamária Gazdag ◽  
Miklós Bodor ◽  
Eszter Berta ◽  
Mónika Katkó ◽  
...  
Keyword(s):  
Clinical Experience ◽  
Clinical Course ◽  
Treatment Protocol ◽  
Treatment Modalities ◽  
Graves Disease ◽  
Novel Treatment ◽  
Graves Orbitopathy ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Novel Therapeutic

Graves’ orbitopathy is the most common extrathyroidal manifestation of Graves’ disease. Up to now, curative treatment modalities for the most severe sight-threatening cases have not been developed. Here the authors summarize the treatment protocol of Graves’ orbitopathy and review novel therapeutic options. They review the literature on this topic and present their own clinical experience. The authors point out that anti-CD20 antibody could positively influence the clinical course of Graves’ orbitopathy. Selenium is efficient in mild cases. Further prospective investigations are warranted. Orv. Hetil., 2014, 155(33), 1295–1300.

scholarly journals Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

2021 ◽  
Vol 7 (8) ◽  
pp. eabd6167
Author(s):  
Capucine L. Grandjean ◽  
Zacarias Garcia ◽  
Fabrice Lemaître ◽  
Béatrice Bréart ◽  
Philippe Bousso
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Critical Path ◽  
B Cell Lymphoma ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Fluorescent Reporter ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

Serum sickness reaction to obinutuzumab in a patient with chronic lymphocytic leukaemia

2021 ◽  
Vol 14 (12) ◽  
pp. e245557
Author(s):  
Oday Elmanaseer ◽  
Alzira R M Avelino ◽  
Amin Azem ◽  
Mihir Raval
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Effective Treatment ◽  
Inflammatory Markers ◽  
Lymphocytic Leukaemia ◽  
Maculopapular Rash ◽  
Serum Sickness ◽  
Rare Entity ◽  
Cd20 Antibody ◽  
Anti Cd20

Serum sickness (SS) is a known phenomenon; however, it is commonly missed due to vague symptoms, and is usually confounded by other aetiologies that present similarly. Obinutuzumab is a novel anti-CD20 antibody agent that has been approved for chronic lymphocytic leukaemia (CLL) treatment. At the time of approval, it was not linked to SS; however, this phenomenon has been recognised with other anti-CD20 agents like rituximab. SS remains a rare entity, but it is important to be recognised accurately and quickly in the appropriate circumstances, so that effective treatment with corticosteroids can be initiated to alleviate inflammatory symptoms. Here we present a patient with CLL who developed maculopapular rash, fever and polyarthritis and elevated inflammatory markers consistent with serum sickness triggered by obinutuzumab and was effectively treated with corticosteroids.

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