Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Ahmadi, Maryam; Mahboudi, Fereidoun; Eidgahi, Mohammad Reza Akbari; Nasr, Reza; Nematpour, Fatemeh; Ahmadi, Samira; Ebadat, Saeedeh; Aghaeepoor, Mojtaba; Davami, Fatemeh

doi:10.1002/btpr.2362

Cited by 14 publications

(11 citation statements)

References 25 publications

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“…Subsequently, antibody fragment was recovered from ptz57R/T vector using ECoRI and XbaI, subcloned into PB513b1 vector digested with ECoRI and XbaI, respectively. Plasmid bearing LC-IRES-HC sequences was previously constructed in our lab [22]. LC-IRES-HC fragment was excised with NheI and NotI and inserted in PB513b1 vector to create CMV-IRES vector [23].…”

Section: Methodsmentioning

confidence: 99%

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Ebadat

Ahmadi

et al. 2017

PLoS ONE

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In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

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Section: Methodsmentioning

confidence: 99%

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Ebadat

Ahmadi

et al. 2017

PLoS ONE

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“…Not only N-pBLIH cells expressed antibody far less than their transposon-based cells, but also their expression levels were negligible in comparison with the other conventionally-created cells, N-pBLPCH and N-pBL2AH, even though a wild-type EMCV-IRES sequence had been incorporated into the donor vector [28]. Moreover, in a parallel study performed in our lab with another enzymatic targeting approach (PhiC31 integrase) using IRES to express a mAb, similar outcomes were observed [29]. …”

Section: Discussionmentioning

confidence: 56%

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

et al. 2017

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Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

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“…Traditional methods for generation of stable cell lines depend on random integration(s) of the transgene that lead to unsteady and unpredictable protein expression. Site-speci c recombination systems are powerful methods for inserting the transgene, which leads to long term and high levels of recombinant protein expression [13]. Among site-speci c recombinases, PhiC31 serine integrase mediates speci c unidirectional integration of the donor plasmid containing transgene in the host genome compared to reversible recombination through bacteriophage recombinases such as Cre and Flp [14,15].…”

Section: Introductionmentioning

confidence: 99%

Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

Ata-Abadi

Dormiani

Varnosfaderani

et al. 2021

Preprint

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PH20 is hyaluronidase that hydrolyze the glycosidic bond of hyaluronic acid as a major proteoglycan found in extracellular matrices. PH20 is used in the subcutaneous space to increase the dispersion and absorption of co-administered drugs. PH20 is also injected against solid tumors for to better penetration of anticancer agents into the tumor tissue and inhibiting the tumor cell growth. In the present study, we have developed HEK293T stable cell lines secreting His-tagged human recombinant PH20 (rhPH20) in the culture supernatant through the PhiC31 integrase system. The produced rhPH20 was quantify using ELISA and turbidimetric assay and its activity was assessed through treatment of mouse cumulus-oocyte-complex (COCs). Furthermore, we have characterized genomic integration of PH20-containing vectors in one of the isolated clone with the highest levels of rhPH20 production. Our results demonstrated that the secreted rhPH20 in the culture supernatant contained a specific activity of approximately 3.5 IU/ml and it was properly able to denote all mouse oocytes. Consequently, it was revealed that PH20-expressing vectors integrated site-specifically in the PhiC31 pseudo attP sites in the host genome. Taken together, these results confirmed successful application of PhiC31 integrase as a robust approach for production of soluble, active rhPH20 in HEK293T cells.

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Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Cited by 14 publications

References 25 publications

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

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