Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Construction of paclitaxel-based antibody–drug conjugates with a PEGylated linker to achieve superior therapeutic index

Dear Editor,The selection of linkers and payloads plays a crucial role in determining the therapeutic indices of antibody–drug conjugates(ADCs).1 Valine–citrulline(Val–Cit)coupled with a self-immolativeρ-aminobenzyl(PAB)spacer as a cleavable dipeptide linker,designated as“VC linker”in this letter,has been popularly used in ADC conjugation.1 However,VC linker is highly hydrophobic2 and,together with hydrophobic payloads at high DARs(drug-toantibody ratios),may turn ADC molecules that have hydrophilic parts in their antibody portions,into aggregation-prone“surfactant-like”structures.

ACE2-targeting monoclonal antibody as potent and broad-spectrum coronavirus blocker

The evolution of coronaviruses,such as SARS-CoV-2,makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after.Here we report a human angiotensin-converting enzyme 2(ACE2)-targeting monoclonal antibody,3E8,blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2,SARS-CoV-2 mutant variants(SARS-CoV-2-D614G,B.1.1.7,B.1.351,B.1.617.1,and P.1).

Fluorescent nanoswitch for monitoring specific pluripotency-related microRNAs of induced pluripotent stem cells： Development of polyethyleneimine- oligonucleotide hybridization probes

The isolation of high-grade （i.e. high-pluripotency） human induced pluripotent stem cells （hiPSCs） is a decisive factor for enhancing the purity of hiPSC populations or differentiation efficiency. A non-invasive imaging system that can monitor microRNA （miRNA） expression provides a useful tool to identify and analyze specific cell populations. However, previous studies on the monitoring/isolation of hiPSCs by miRNA expression have limited hiPSCs＇ differentiation system owing to long-term incubation with miRNA imaging probe-nanocarriers. Therefore, we focused on monitoring high-grade hiPSCs without influencing the pluripotency of hiPSCs. We reduced nanoparticle transfection time, because hiPSCs are prone to spontaneous differentiation under external factors during incubation. The fluorescent nanoswitch （＂ON＂ with target miRNA）, which can be applied for either imaging or sorting specific cells by fluorescence signals, contains an miRNA imaging probe （miP） and a PEI-PEG nanoparticle （miP-P）. Consequently, this nanoswitch can sense various endogenous target miRNAs within 30 min in vitro, and demonstrates strong potential for not only imaging but also sorting pluripotent hiPSCs without affecting pluripotency. Moreover, miP-P-treated hiPSCs differentiate well into endothelial cells, indicating that miP-P does not alter the pluripotency of hiPSCs. We envisage that this miRNA imaging system could be valuable for identifying and sorting high-grade hiPSCs for improved practical applications.

Transcriptomics Curation of SARS-CoV-2 Related Host Genes in Mice With COVID-19 Comorbidity:A Pilot Study

The pandemic of coronavirus disease 2019(COVID-19),a respiratory disease caused by a novel severe acute respiratory syndrome coronavirus-2,is causing substantial morbidity and mortality.Along with the respiratory symptoms,underlying diseases in senior patients,such as diabetes,hypertension,and coronary heart disease,are the most common comorbidities,which cause more severe outcomes and even death.During cellular attachment and entry of severe acute respiratory syndrome coronavirus-2,the key protein involved is the angiotensin I converting enzyme 2(ACE2),which is located on the membrane of host cells.Here,we aim to curate an expression profile of Ace2 and other COVID-19 related genes across the available diabetes murine strains.Based on strictly manual curation and bioinformatics analysis of the publicly deposited expression datasets,Ace2 and other potentially involved genes such as Furin,Tmprss2,Ang,and Ang2 were examined.We found that Ace2 expression is rather ubiquitous in three selected diabetes prone strains(db/db,ob/ob and diet-induced obese).With the most abundant datasets present,the liver shows a medium Ace2 expression level compared with the lungs,pancreatic islets,brain and even T cells.Age is a more critical factor for Ace2 expression in db/db compared with the other two strains.Besides Ace2,the other four host genes showed varied levels of correlation to each other.To accelerate research on the interaction between COVID-19 and underlying diseases,the Murine4Covid transcriptomics database(www.geneureka.org/Murine4Covid)will facilitate the design of research on COVID-19 and comorbidities.