Production of “biobetter” glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

Al-Qahtani, Alanod; Al-Mansoori, Layla; Bashraheel, Sara S.; Rashidi, Fatma B.; Al-Yafei, Afrah; Elsinga, Philip H.; Dömling, Alexander; Goda, Sayed K.

doi:10.1016/j.ejps.2018.10.014

Cited by 21 publications

(8 citation statements)

References 29 publications

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“…In vitro serum stability of the produced PEGylated CPG2 fusion proteins’ catalytic activity was examined at physiologic conditions. In agreement with our previous results, 10 there was a trend of improvement in the enzyme stability of PEG-WT compared with non-PEGylated WT-CPG2 ( Figure 4 ). Interestingly, both single and double CPG2 fusion proteins (PEG X-CPG2 and PEG X-CPG2-X) stability was not significantly affected following PEGylation.…”

Section: Resultssupporting

confidence: 93%

“… 24 These conformational changes might explain the lower activity of the PEGylated double-fused CPG2 ( Figure 4 ), also as shown in our previous studies. 10 …”

Section: Discussionmentioning

confidence: 99%

“… 21 Two different studies found that conjugation of CPG2 either with the fusion peptide (CNGRC) or PEG polymer formed fusion proteins and PEGylated CPG2, respectively with higher serum stability and lower ex vivo immunotoxicity compared with the nonfused or non-PEGylated CPG2. 10 , 18 In this study, we produced new PEGylated fused CPG2 proteins which tested for their stability and immunotoxicity. Our data showed that the serum stability of the PEGylated CPG2 fusion proteins was not changed in relation to the non-PEGylated ones ( Figure 4 ).…”

Section: Discussionmentioning

confidence: 99%

“…Prepared samples (about 70 µL of 10 µM) concentration were placed in a SUPRASIL Quartz cuvette demountable rectangular “.2 mm light-path length” (Hellma®). The applied CD parameters were as previously mentioned, 10 band width 1 nm, scan time/point of .5 s, and for each sample 4 scans were taken, which were averaged and smoothed using the CD analysis software. The produced spectra were subtracted from an averaged CD spectrum of the used blank (Milli Q water) baseline.…”

Section: Methodsmentioning

confidence: 99%

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Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

Al-Mansoori

Qahtani

Elsinga

et al. 2021

Technol Cancer Res Treat

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Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC–carboxypeptidase G2 (CPG2) and CNGRC–CPG2–CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC–CPG2, and CNGRC–CPG2–CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug “ZD2767P” cytotoxic effect in association with PEG CPG2–CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC–CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

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Section: Resultssupporting

confidence: 93%

“… 24 These conformational changes might explain the lower activity of the PEGylated double-fused CPG2 ( Figure 4 ), also as shown in our previous studies. 10 …”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

Al-Mansoori

Qahtani

Elsinga

et al. 2021

Technol Cancer Res Treat

Self Cite

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“…Although its stability in the blood is high enough to eliminate high concentrations of blood MTX, this might not be enough for successful application in ADEPT [75]. Therefore, several strategies have been applied to increase the resistance of glucarpidase to proteolytic enzymes, such as PEGylation, fusion with human serum albumin [76,77], and circular permutations [75].…”

Section: Resultsmentioning

confidence: 99%

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Behzadipour

Hemmati

2019

Molecules

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Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

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Biological Implications of Polyethylene Glycol and PEGylation: Therapeutic Approaches Based on Biophysical Studies and Protein Structure-Based Drug Design Tools

Raina

Singh

Islam

2021

Innovations and Implementations of Computer Aided Drug Discovery Strategies in Rational Drug Design

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Production of “biobetter” glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

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Cited by 21 publications

References 29 publications

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Biological Implications of Polyethylene Glycol and PEGylation: Therapeutic Approaches Based on Biophysical Studies and Protein Structure-Based Drug Design Tools

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