Circulating microRNAs (miRNAs) are a group of small RNA molecules that have been shown to be involved in several human diseases, such as cancers, neurodegenerative disease, and inflammation. Their sequences are highly conserved across species. miRNAs are present in biological fluids, are differentially expressed and can be used as biomarkers reported to be translational for research and drug development as improved diagnostic assays. Multiple techniques used to measure miRNA expression in biological fluids are labor-intensive and subject to variability. The objective of this study was to develop an extraction-free and amplification-free detection method using Single Molecule Counting (SMC™) technology and the SMCxPRO™ instrument combined with dynamic chemical labelling (DCL) chemistry by DESTINA for the direct detection of microRNAs in biological samples. As a proof of concept, miR-122 (miR-122-5p), a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI) has been selected. A DESTINA abasic probe for detecting miR-122 is conjugated to superparamagnetic beads. These beads are added to DILI patient serums treated with DESTINA Stabiltech and  incubated with a biotinylated DESTINA SMART Nucleobase. Following miR-122 target molecule hybridization to the DESTINA probe, the reactive SMART nucleobase is then covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. The biotin-labeled superparamagnetic beads are then incubated with Alexa Fluor®647-conjugated streptavidin and the labeled heteroduplex complex is cleaved from the superparamagnetic beads before being detected by the SMCxPRO™instrument. This is the first example where SMC™ technology has been used for molecular applications, allowing rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for research.

The AIDS epidemic is a global challenge with 38 million people living with HIV and 1.7 million new infections along with 690K deaths in 2019. To date, HIV-1 vaccine clinical trials have not been successful in eliciting robust broad neutralizing antibody responses that can prevent infections. Alternative approaches, such as passive immunization with broadly neutralizing monoclonal antibodies, are being considered to help prevent new HIV infections. VRC01, an HIV-gp120 CD4bs directed potent and broadly neutralizing antibody (bNAb), which neutralizes about 91% of HIV-1 strains of a panel consisting of 190 Env-pseudotyped viral strains with a half max inhibitory concentration (IC50) of <50 ug/mL, is being tested for safety and efficacy in preventing HIV infection. Since the majority of HIV-1 transmissions occur through mucosal routes, it is essential to quantify the levels of the passively administered bNAb in mucosal samples. This helps to determine the levels of the bNAbs that have localized to mucosal sites preventing infection by neutralizing the virus. Traditional ELISAs and other immunoassays with a lower limit of quantification (LLOQ) in the range of 1 ug/mL are not sensitive enough to accurately quantify the low levels of VRC01 in mucosal samples. Hence, we sought to develop a custom ultra-sensitive Single Molecule Counting (SMC™) magnetic microparticle-based immunoassay with a greater than 4-log linear dynamic range (LDR) and LLOQ less than 120 pg/mL. We successfully quantified VRC01 levels in rectal, cervical and oral mucosal samples in two passive immunization studies conducted with VRC01- VRC 601 and VRC 602. The assay was able to successfully quantify VRC01 levels in mucosal samples from all dosage groups from these trials. This accurate and sensitive immunoassay can be utilized in vaccine research for quantifying low antibody levels in mucosal and other sample types in the context of HIV-1 pre-clinical and clinical vaccine trials.

First in human (FIH) clinical trials of bispecific immunostimulatory antibodies commonly require a low starting dose based on a minimum anticipated biological effect level (MABEL) approach.  To quantify serum drug concentrations at low dose levels, an ultrasensitive pharmacokinetic (PK) assay is required for Aptevo’s bispecific antibody, ADAPTIR-X. The SMCxPRO™ platform was identified as potentially having the capability to reach the required assay sensitivity.   A feasibility assessment was undertaken utilizing MilliporeSigma’s Custom Assay & Sample Testing (CAST) team and the custom assay development SMCxPLORE™ option, including testing of sensitivity, matrix effects, accuracy, and precision.  From initial discussions to sample analysis, Aptevo and the SMC™ team efficiently collaborated to produce high quality, informative data, leading to the purchase and implementation of SMCxPRO™ technology at Aptevo.  This presentation will introduce the CAST team and their in-house custom assay services, as well as describe the steps taken during the SMCxPLORE™ feasibility assessment to achieve a sensitivity of ~0.32 pg/mL for the ADAPTIR-X PK assay.

Advances in bioanalytical tools has enabled the evaluation of appropriate biomarkers as well as assess drug efficacy and drug safety facilitating both translational and clinical research.  The accurate detection and monitoring of clinically relevant low-level biomarkers, such as, cTnI, IL-17a, IL-17F, and IL-23 can provide valuable insights into early disease progression or indication of disease and thus have become attractive candidates for targeted therapies of pharmacokinetic and pharmacodynamic (PK/PD) studies. However, due to the low-abundance of these biomarkers, obtaining robust measurements in clinical samples has been difficult. Furthermore, as therapeutics become more complex the modality induced immune responses observed during clinical trials, particularly the early incidence of anti-drug antibodies (ADA) induction and the implications of these responses’ in product safety and efficacy, is crucial for any drug development program. The recent FDA guidance (Jan 2019) recommends 100 ng/mL sensitivity for clinical screening and confirmatory assays events as well as the necessity of these assays to be target and drug tolerant, the latter of which can often be a challenge.  This presentation will discuss how research studies, including PK/PD and ADA, using Single Molecule Counting (SMC™) Technology, have enabled researchers to gain unprecedented insights into complex disease processes , drug safety and efficacy as well as potentially overcome some of the challenges to facilitate the drug development program.