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.

Human platelet lysates for human cell propagation.

A pathogen-free and standardized xeno-free supplement of growth media is required for the ex vivo propagation of human cells used as advanced therapeutic medicinal products and for clinical translation in regenerative medicine and cell therapies. Human platelet lysate (HPL) made from therapeutic-grade platelet concentrate (PC) is increasingly regarded as being an efficient xeno-free alternative growth medium supplement to fetal bovine serum (FBS) for clinical-grade isolation and/or propagation of human cells. Most experimental studies establishing the superiority of HPL over FBS were conducted using mesenchymal stromal cells (MSCs) from bone marrow or adipose tissues. Data almost unanimously concur that MSCs expanded in a media supplemented with HPL have improved proliferation, shorter doubling times, and preserved clonogenicity, immunophenotype, in vitro trilineage differentiation capacity, and T-cell immunosuppressive activity. HPL can also be substituted for FBS when propagating MSCs from various other tissue sources, including Wharton jelly, the umbilical cord, amniotic fluid, dental pulp, periodontal ligaments, and apical papillae. Interestingly, HPL xeno-free supplementation is also proving successful for expanding human-differentiated cells, including chondrocytes, corneal endothelium and corneal epithelium cells, and tenocytes, for transplantation and tissue-engineering applications. In addition, the most recent developments suggest the possibility of successfully expanding immune cells such as macrophages, dendritic cells, and chimeric antigen receptor-T cells in HPL, further broadening its use as a growth medium supplement. Therefore, strong scientific rationale supports the use of HPL as a universal growth medium supplement for isolating and propagating therapeutic human cells for transplantation and tissue engineering. Efforts are underway to ensure optimal standardization and pathogen safety of HPL to secure its reliability for clinical-grade cell-therapy and regenerative medicine products and tissue engineering.

Measurement of Pre-Existing IgG and IgM Antibodies against Polyethylene Glycol in Healthy Individuals.

Polyethylene glycol (PEG) is a biocompatible polymer that is often attached to therapeutic molecules to improve bioavailability and therapeutic efficacy. Although antibodies with specificity for PEG may compromise the safety and effectiveness of PEGylated medicines, the prevalence of pre-existing anti-PEG antibodies in healthy individuals is unclear. Chimeric human anti-PEG antibody standards were created to accurately measure anti-PEG IgM and IgG antibodies by direct ELISA with confirmation by a competition assay in the plasma of 1504 healthy Han Chinese donors residing in Taiwan. Anti-PEG antibodies were detected in 44.3% of healthy donors with a high prevalence of both anti-PEG IgM (27.1%) and anti-PEG IgG (25.7%). Anti-PEG IgM and IgG antibodies were significantly more common in females as compared to males (32.0% vs 22.2% for IgM, p < 0.0001 and 28.3% vs 23.0% for IgG, p = 0.018). The prevalence of anti-PEG IgG antibodies was higher in younger (up to 60% for 20 year olds) as opposed to older (20% for >50 years) male and female donors. Anti-PEG IgG concentrations were negatively associated with donor age in both females (p = 0.0073) and males (p = 0.026). Both anti-PEG IgM and IgG strongly bound PEGylated medicines. The described assay can assist in the elucidation of the impact of anti-PEG antibodies on the safety and therapeutic efficacy of PEGylated medicines.

Versatile online SPE-HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials.

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide. Sample clean-up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH2 PO4 pH 2.9. Online transfer to an analytical µBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH2 PO4 pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN-38 glucuronide and Irinotecan or 540 nm for SN-38 results in high sensitivity (1-2 pg) and short (∼10 min) run times. The method was used to determine the degree of SN-38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN-38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan-based tumor targeting therapies.

Reversible glycosidic switch for secure delivery of molecular nanocargos.

Therapeutic drugs can leak from nanocarriers before reaching their cellular targets. Here we describe the concept of a chemical switch which responds to environmental conditions to alternate between a lipid-soluble state for efficient cargo loading and a water-soluble state for stable retention of cargos inside liposomes. A cue-responsive trigger allows release of the molecular cargo at specific cellular sites. We demonstrate the utility of a specific glycosidic switch for encapsulation of potent anticancer drugs and fluorescent compounds. Stable retention of drugs in liposomes allowed generation of high tumor/blood ratios of parental drug in tumors after enzymatic hydrolysis of the glycosidic switch in the lysosomes of cancer cells. Glycosidic switch liposomes could cure mice bearing human breast cancer tumors without significant weight loss. The chemical switch represents a general method to load and retain cargos inside liposomes, thereby offering new perspectives in engineering safe and effective liposomes for therapy and imaging.

Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy.

Triple-negative breast cancer (TNBC) lacks effective treatment options due to the absence of traditional therapeutic targets. The epidermal growth factor receptor (EGFR) has emerged as a promising target for TNBC therapy because it is overexpressed in about 50% of TNBC patients. Here we describe a PEG engager that simultaneously binds polyethylene glycol and EGFR to deliver PEGylated nanomedicines to EGFR+ TNBC. The PEG engager displays conditional internalization by remaining on the surface of TNBC cells until contact with PEGylated nanocarriers triggers rapid engulfment of nanocargos. PEG engager enhances the anti-proliferative activity of PEG-liposomal doxorubicin to EGFR+ TNBC cells by up to 100-fold with potency dependent on EGFR expression levels. The PEG engager significantly increases retention of fluorescent PEG probes and enhances the antitumour activity of PEGylated liposomal doxorubicin in human TNBC xenografts. PEG engagers with specificity for EGFR are promising for improved treatment of EGFR+ TNBC patients.

Engineering Chimeric Receptors To Investigate the Size- and Rigidity-Dependent Interaction of PEGylated Nanoparticles with Cells.

Attachment of ligands to the surface of nanoparticles (NPs) is an attractive approach to target specific cells and increase intracellular delivery of nanocargos. To expedite investigation of targeted NPs, we engineered human cancer cells to express chimeric receptors that bind polyethylene glycol (PEG) and internalize stealth NPs in a fashion similar to ligand-targeted liposomes against epidermal growth factor receptor 1 or 2 (HER1 or HER2), which are validated targets for cancer therapy. Measurement of the rate of endocytosis and lysosomal accumulation of small (80-94 nm) or large (180-220 nm) flexible liposomes or more rigid lipid-coated mesoporous silica particles in human HT29 colon cancer and SKBR3 breast cancer cells that express chimeric receptors revealed that larger and more rigid NPs were internalized more slowly than smaller and more flexible NPs. An exception is when both the small and large liposomes underwent endocytosis via HER2. HER1 mediated faster and greater uptake of NPs into cells but retained NPs less well as compared to HER2. Lysosomal accumulation of NPs internalized via HER1 was unaffected by NP rigidity but was inversely related to NP size, whereas large rigid NPs internalized by HER2 displayed increased lysosomal accumulation. Our results provide insight into the effects of NP properties on receptor-mediated endocytosis and suggest that anti-PEG chimeric receptors may help accelerate investigation of targeted stealth NPs.

Selective Delivery of PEGylated Compounds to Tumor Cells by Anti-PEG Hybrid Antibodies.

Polyethylene glycol (PEG) is attached to many peptides, proteins, liposomes, and nanoparticles to reduce their immunogenicity and improve their pharmacokinetic and therapeutic properties. Here, we describe hybrid antibodies that can selectively deliver PEGylated medicines, imaging agents, or nanomedicines to target cells. Human IgG1 hybrid antibodies αPEG:αHER2 and αPEG:αCD19 were shown by ELISA, FACS, and plasmon resonance to bind to both PEG and HER2 receptors on SK-BR-3 breast adenocarcinoma and BT-474 breast ductal carcinoma cells or CD19 receptors on Ramos and Raji Burkitt's lymphoma cells. In addition, αPEG:αHER2 specifically targeted PEGylated proteins, liposomes, and nanoparticles to SK-BR-3 cells that overexpressed HER2, but not to HER2-negative MCF-7 breast adenocarcinoma cells. Endocytosis of PEGylated nanoparticles into SK-BR-3 cells was induced specifically by the αPEG:αHER2 hybrid antibody, as observed by confocal imaging of the accumulation of Qdots inside SK-BR-3 cells. Treatment of HER2(+) SK-BR-3 and BT-474 cancer cells with αPEG:αHER2 and the clinically used chemotherapeutic agent PEGylated liposomal doxorubicin for 3 hours enhanced the in vitro effectiveness of PEGylated liposomal doxorubicin by over two orders of magnitude. Hybrid anti-PEG antibodies offer a versatile and simple method to deliver PEGylated compounds to cellular locations and can potentially enhance the therapeutic efficacy of PEGylated medicines.