scholarly journals Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

2021 ◽  
Author(s):  
Fatemeh Khademi ◽  
Pantea Mohammadi ◽  
Ali Mostafaei
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Viability ◽  
B Cell Lymphoma ◽  
Chimeric Antibody ◽  
Chimeric Mouse ◽  
Chain Reaction ◽  
Chimeric Antibodies ◽  
Overlap Extension ◽  
Polymerase Chain ◽  
Anti Cd20

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Mantle Cell Lymphoma

1999 ◽  
Vol 123 (12) ◽  
pp. 1182-1188 ◽  
Author(s):  
Rebecca C. Hankin ◽  
Susan V. Hunter
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Diagnostic Tests ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Mantle Cell ◽  
Polymerase Chain

Abstract Objective.—This article summarizes the most useful ancillary immunohistochemical and molecular assays for use in the diagnosis of mantle cell lymphoma. Data Sources.—The English language literature was surveyed, with an emphasis on recent publications, for articles presenting key advances in the molecular characterization of mantle cell lymphomas and for series of cases testing the utility of molecular diagnostic tests. The authors' series of 26 small B-cell lymphomas, analyzed for the cyclin D1 protein by paraffin immunohistochemistry and for t(11;14) by polymerase chain reaction, is included. Conclusions.—Mantle cell lymphoma, a B-cell lymphoma now recognized in the 1994 Revised European-American Classification of Lymphoid Neoplasms (REAL) classification, is a relatively aggressive lymphoma with a poor prognosis. Its characteristic t(11;14)(q13;q32) translocation has a role in oncogenesis and has been exploited for molecular diagnostic tests, but these tests vary in sensitivity, specificity, and ease of use. Improved immunohistochemical tests are sufficient to confirm the diagnosis in most cases. Conventional cytogenetics and molecular diagnostic tests for t(11;14)—Southern blot and polymerase chain reaction analysis—may be helpful in selected cases, but are laborious or of limited sensitivity. Other methods, such as fluorescence in situ hybridization, need further development to provide faster, more sensitive diagnosis.

Extranodal Marginal Zone B-Cell Lymphoma Genotyping byAlu— Polymerase Chain Reaction

2000 ◽  
Vol 38 (5-6) ◽  
pp. 605-610
Author(s):  
Francesco Bertoni ◽  
Roman Müllenbach ◽  
Massimo Broggini ◽  
Enrico Roggero ◽  
Franco Cavalli ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Chain Reaction ◽  
Polymerase Chain

Creating Insertions or Deletions Using Overlap Extension Polymerase Chain Reaction (PCR) Mutagenesis

2018 ◽  
Vol 2018 (8) ◽  
pp. pdb.prot097758 ◽  
Author(s):  
Matteo Forloni ◽  
Alex Y. Liu ◽  
Narendra Wajapeyee
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Overlap Extension ◽  
Polymerase Chain

scholarly journals Cloning recombinant consensus interferon with overlap extension polymerase chain reaction

2008 ◽  
Vol 16 (5) ◽  
pp. 479
Author(s):  
Shun-Ai Liu ◽  
Ryutaro Asano ◽  
Jing-Jing Guo ◽  
Zhi-Ying Liu ◽  
Kang-Kang Yu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Consensus Interferon ◽  
Overlap Extension ◽  
Polymerase Chain

scholarly journals Effects of Decitabine and Homoharringtonine in Combination in Leukemia Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5229-5229
Author(s):  
Suxia Geng ◽  
Han Yao ◽  
Jianyu Weng ◽  
Xin Huang ◽  
Ping Wu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Viability ◽  
Cell Lines ◽  
Caspase 3 ◽  
Synergistic Effects ◽  
Apoptosis Induction ◽  
Caspase 9 ◽  
Chain Reaction ◽  
Mrna And Protein Expression ◽  
Polymerase Chain

Abstract Background: The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates for overall response and complete remission. We aimed to investigate the effects of the combination of DAC and homoharringtonine (HHT) in the SKM-1 and Kg-1a cell lines and explore their associated mechanisms. Methods: Cell viability was estimated by MTS assay. Cell apoptosis were detected by flow cytometry analysis and PI/Annexin V staining. Western blot and quantitative reverse transcription-polymerase chain reaction assays were performed to detect the expression of apoptosis-related genes including caspase-3, caspase-9, BCL-2 and BCL-XL and DNA methyltransferases(DNMT) including DNMT1, DNMT3A and DNMT3B. Changes in LINE-1 gene methylation were assessed by quantitative polymerase chain reaction after bisulfite conversion. The effects of the combinations were estimated using CalcuSyn software. Results: The combination of DAC and HHT showed synergistic effects for inhibiting cell viability in SKM-1 and KG-1a cell lines. This combination can enhance inhibition of colony formation and apoptosis induction of DAC alone in SKM-1 cell line. However, in Kg-1a cells, this combination only enhanced the apoptosis induction of DAC alone. For SKM-1 cells, further study found that different doses of DAC and HHT alone have different effects on the expression of the apoptosis-related genes caspase-3, caspase-9, BCL-XL and BCL-2. The combination of 0.4 μM DAC and 5 nM HHT treatment significantly increased the mRNA expression of caspase-3 (P=0.0005) and caspase-9 (P=0.0075) and decreased the expression of BCL-2 (P=0.0331), but has no significantly effect on the BCL-XL (P=0.3436) compared with 0.4 μM DAC alone. However, 4 μM DAC plus 50 nM HHT had no significant effects on the mRNA and protein expression of the apoptosis-related genes examined compared with 4 μM DAC alone (P>0.05). Low-dose DAC induced greater hypomethylation than higher doses of the drug. Whereas HHT had no demethylation effects, and also had no enhancement effects with DAC on the hypomethylation and mRNA and protein expression of DNMT1, DNMT3A and DNMT3B in SKM-1 cells. Conclusion: The combination of DAC and HHT has synergistic effects on cell viability inhibition and enhancement effects on cell apoptosis in SKM-1 and KG-1a cell lines. But this combination did not enhance the hypomethylation effect of DAC.These data suggest that DAC used in combination with HHT has clinical potential in the treatment of MDS and AML. Disclosures No relevant conflicts of interest to declare.

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