A competitive ligand-binding assay to detect neutralizing antibodies to a bispecific drug using a multiplex Meso Scale Discovery platform.

Background: Monitoring appearance of neutralizing antibodies (NAbs) to multidomain large molecule drugs is a challenging task. Materials & methods: Here, we report development of a competitive ligand-binding assay for detection of NAbs to a bispecific candidate drug using a multiplex Meso Scale Discovery platform, which allows for detection of NAbs to both drug arms in the same sample. Results: The assay has sensitivity better than 250 ng/ml and is tolerant to the presence of drug at concentration >600 µg/ml and to the level of soluble target(s) >400 ng/ml. Conclusion: Our data suggest that multiplex approach can be successfully used for development of NAb assays in competitive ligand-binding assay format.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

An Assessment of LRRK2 Serine 935 Phosphorylation in Human Peripheral Blood Mononuclear Cells in Idiopathic Parkinson's Disease and G2019S LRRK2 Cohorts.

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.

A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers.

PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

A Multi-site In-depth Evaluation of the Quanterix Simoa from a User's Perspective.

An in-depth evaluation of the Quanterix© Simoa™ platform was undertaken by scientists from the AAPS Emerging Technologies Focus Group to determine the overall performance of the technology as well as provide guidance to future users. In order to test the platform in a non-GLP bioanalytical setting, a cross-site evaluation of the Quanterix IL-6 biomarker kit was performed. Parameters tested during this evaluation included sensitivity, accuracy and precision, and parallelism in human serum from normal individuals. The results demonstrated improved sensitivity compared to the claimed sensitivity of other commercially available IL-6 kits and showed excellent site-to-site reproducibility. Observed issues included difficulties with system reliability and a lack of parallelism and specificity in a subset of samples. Overall, these results demonstrate that while there are challenges to the Simoa platform this technology offers automation capabilities and excellent sensitivity that enhance bioanalysis especially of low-abundance analytes.

One mouse, one pharmacokinetic profile: quantitative whole blood serial sampling for biotherapeutics.

PURPOSE: The purpose of this study was to validate the approach of serial sampling from one mouse through ligand binding assay (LBA) quantification of dosed biotherapeutic in diluted whole blood to derive a pharmacokinetic (PK) profile. METHODS: This investigation compared PK parameters obtained using serial and composite sampling methods following administration of human IgG monoclonal antibody. The serial sampling technique was established by collecting 10 µL of blood via tail vein at each time point following drug administration. Blood was immediately diluted into buffer followed by analyte quantitation using Gyrolab to derive plasma concentrations. Additional studies were conducted to understand matrix and sampling site effects on drug concentrations. RESULTS: The drug concentration profiles, irrespective of biological matrix, and PK parameters using both sampling methods were not significantly different. There were no sampling site effects on drug concentration measurements except that concentrations were slightly lower in sodium citrated plasma than other matrices. CONCLUSIONS: We recommend the application of mouse serial sampling, particularly with limiting drug supply or specialized animal models. Overall the efficiencies gained by serial sampling were 40-80% savings in study cost, animal usage, study length and drug conservation while inter-subject variability across PK parameters was less than 30%.

Quantitative analysis of target coverage and germinal center response by a CXCL13 neutralizing antibody in a T-dependent mouse immunization model.

PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.

Bioanalytical platform comparison using a generic human IgG PK assay format.

A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG1 monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte, and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance. Assay parameters such as sensitivity, dynamic range, minimum required dilution and sample volume as well as other considerations such as per-run cost, technology availability, requisite equipment and necessary reagent modifications were evaluated toward the determination of a default go-to assay platform for monoclonal antibody biotherapeutics in this laboratory. Based primarily on superior assay performance, Meso Scale Discovery and Gyros were selected from the four technologies evaluated as our default platforms for non-regulated (discovery) study support. As an adjunct, immunoaffinity LC-MS/MS was explored as an alternate platform for generic Fc quantitation and was found to perform similarly to the ligand-binding assays.

Pharmacokinetic, biodistribution, and biophysical profiles of TNF nanobodies conjugated to linear or branched poly(ethylene glycol).

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.