Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

Accelerated clearance by antibodies against methoxy PEG depends on pegylation architecture.

Methoxy polyethylene glycol (mPEG) is attached to many proteins, peptides, nucleic acids and nanomedicines to improve their biocompatibility. Antibodies that bind PEG are present in many individuals and can be generated upon administration of pegylated therapeutics. Anti-PEG antibodies that bind to the PEG "backbone" can accelerate drug clearance and detrimentally affect drug activity and safety, but no studies have examined how anti-methoxy PEG (mPEG) antibodies, which selectively bind the terminus of mPEG, affect pegylated drugs. Here, we investigated how defined IgG and IgM monoclonal antibodies specific to the PEG backbone (anti-PEG) or terminal methoxy group (anti-mPEG) affect pegylated liposomes or proteins with a single PEG chain, a single branched PEG chain, or multiple PEG chains. Large immune complexes can be formed between all pegylated compounds and anti-PEG antibodies but only pegylated liposomes formed large immune complexes with anti-mPEG antibodies. Both anti-PEG IgG and IgM antibodies accelerated the clearance of all pegylated compounds but anti-mPEG antibodies did not accelerate clearance of proteins with a single or branched PEG molecule. Pegylated liposomes were primarily taken up by Kupffer cells in the liver, but both anti-PEG and anti-mPEG antibodies directed uptake of a heavily pegylated protein to liver sinusoidal endothelial cells. Our results demonstrate that in contrast to anti-PEG antibodies, immune complex formation and drug clearance induced by anti-mPEG antibodies depends on pegylation architecture; compounds with a single or branched PEG molecule are unaffected by anti-mPEG antibodies but are increasingly affected as the number of PEG chain in a structure increases.

Pathway analysis of smoking-induced changes in buccal mucosal gene expression.

Background: Cigarette smoking is the leading preventable cause of death worldwide, and it is the most common cause of oral cancers. This study aims to provide a deeper understanding of the molecular pathways in the oral cavity that are altered by exposure to cigarette smoke. Methods: The gene expression dataset (accession number GSE8987, GPL96) of buccal mucosa samples from smokers (n = 5) and never smokers (n = 5) was downloaded from The National Center for Biotechnology Information's (NCBI) Gene Expression Omnibus (GEO) repository. Differential expression was ascertained via NCBI's GEO2R software, and Ingenuity Pathway Analysis (IPA) software was used to perform a pathway analysis. Results: A total of 459 genes were found to be significantly differentially expressed in smoker buccal mucosa (p < 0.05). A total of 261 genes were over-expressed while 198 genes were under-expressed. The top canonical pathways predicted by IPA were nitric oxide and reactive oxygen production at macrophages, macrophages/fibroblasts and endothelial cells in rheumatoid arthritis, and thyroid cancer pathways. The IPA upstream analysis predicted that the TP53, APP, SMAD3, and TNF proteins as well as dexamethasone drug would be top transcriptional regulators. Conclusions: IPA highlighted critical pathways of carcinogenesis, mainly nitric oxide and reactive oxygen production at macrophages, and confirmed widespread injury in the buccal mucosa due to exposure to cigarette smoke. Our findings suggest that cigarette smoking significantly impacts gene pathways in the buccal mucosa and may highlight potential targets for treating the effects of cigarette smoking. Supplementary Information: The online version contains supplementary material available at 10.1186/s43042-022-00268-y.

Transient AID expression for in situ mutagenesis with improved cellular fitness.

Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3-5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.