A novel biomarker assay qualification strategy for rare human matrices - a case study of biomarkers in aqueous humor.

Background: Measuring pharmacodynamic biomarkers near the therapeutic site of action presents considerable challenges for sites with limited matrix volume or difficult access. Bioanalytical method qualification requires the use of numerous matrix samples, which is problematic for rare matrices. The aim of this study was to design and implement a streamlined, fit-for-purpose strategy for qualification of biomarker assays in rare matrices. Materials & methods: A multiplexed biomarker immunoassay was developed in human aqueous humor. Results: Our strategy was successfully implemented, providing characterization of assay performance while reducing number of samples in assay qualification. Our assay was used in clinical trial support for an ophthalmic drug candidate. Conclusion: Our results indicate this approach can be applied to other early stage drug development programs facing similar challenges.

Enrichment of circulating tumor-derived extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 µL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology.

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.

Single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties.

Bispecific antibody production using single host cells has been a new advancement in the antibody engineering field. We previously showed comparable in vitro biological activity and in vivo mouse pharmacokinetics (PK) for two novel single cell variants (v10 and v11) and one traditional dual cell in vitro-assembled anti-human epidermal growth factor receptor 2/CD3 T-cell dependent bispecific (TDB) antibodies. Here, we extended our previous work to assess single cell-produced bispecific variants of a novel TDB against FcRH5, a B-cell lineage marker expressed on multiple myeloma (MM) tumor cells. An in vitro-assembled anti- FcRH5/CD3 TDB antibody was previously developed as a potential treatment option for MM. Two bispecific antibody variants (designs v10 and v11) for manufacturing anti-FcRH5/CD3 TDB in single cells were compared to in vitro-assembled TDB in a dual-cell process to understand whether differences in antibody design and production led to any major differences in their in vitro biological activity, in vivo mouse PK, and PK/pharmacodynamics (PD) or immunogenicity in cynomolgus monkeys (cynos). The binding, in vitro potencies, in vitro pharmacological activities and in vivo PK in mice and cynos of these single cell TDBs were comparable to those of the in vitro-assembled TDB. In addition, the single cell and in vitro-assembled TDBs exhibited robust PD activity and comparable immunogenicity in cynos. Overall, these studies demonstrate that single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties, and support further development of single-cell production method for anti-FcRH5/CD3 TDBs and other single-cell bispecifics.

Fit-for-purpose biomarker immunoassay qualification and validation: three case studies.

AIM: To improve on the efficiency of biomarker assay readiness, and for reliable biomarker data to support three drug programs, we implemented a fit-for-purpose approach, qualifying two biomarker assays and validating a third. Results/methodology: The qualification strategy and selection of experiments for two exploratory biomarkers (CXCL1, CCL19) was determined by the intended use of the biomarker data. The third biomarker, IL-6, was validated as the data would be used in monitoring patient safety during dose-escalation studies in a Phase I trial. All three assays passed a priori acceptance criteria. CONCLUSION: These assays highlight strategies and methodologies for a fit-for-purpose approach. Minimum qualification, full qualification and validation were chosen and supported programs at different stages of drug development.

Commercial biomarker assays: friend and foe.

AIM: Commercial kits can provide a convenient solution for measuring circulating biomarkers to support drug development. However, their suitability should be assessed in the disease matrix of interest. METHODOLOGY: Twelve biomarkers were evaluated in samples from patients with rheumatoid arthritis. We used immunoassay kits from five vendors on three multiplexed platforms. Kit suitability was evaluated on the basis of detectability and prespecified performance acceptance criteria. RESULTS: Assays had varying levels of sensitivity and susceptibility to interference by matrix components. Only a few assays in the multiplexed kits were found to be suitable. In general, kits for analytes that passed our assay criteria showed good correlation between vendors. CONCLUSION: The data from this study demonstrate that the majority of assays on multiplexed kits evaluated either lacked sensitivity and/or had poor performance, which diminishes the utility of the multiplexing approach.