Monoclonal antibody therapies are an important approach for the treatment of hematologic malignancies, but typically show low single‐agent activity. Bispecific antibodies, however, redirect immune cells to the tumor for subsequent lysis, and preclinical and accruing clinical data support single‐agent efficacy of these agents in hematologic malignancies, presaging an exciting era in the development of novel bispecific formats. This review discusses recent developments in this area, highlighting the challenges in delivering effective immunotherapies for patients.

The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on ontarget elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

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There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.

Abstract. The purpose of this manuscript is to provide a summary of the evaluation done by the Throughput and Multiplexing subteam on five emerging technologies: Single molecule array (Simoa™), Optimiser™, CyTOF® (Mass cytometry), SQIDLite™, and iLite™. Most of the information is presented with a minimum amount of published data and much is based on discussions with users and the vendor, to help provide the reader with an unbiased assessment of where the subteam sees each technology fitting best in the bioanalysis of large molecules. The evaluation focuses on technologies with advantages in throughput and multiplexing, but is wide enough to capture their strengths in other areas. While all platforms may be suited to support bioanalysis in the discovery space, because of their emergent nature, only Optimiser and SQIDLite are currently ready to be used in the regulated space. With the exception of Optimiser, each instrument/technology requires an up-front investment from the bioanalytical lab that will need justification during capital budget discussions. Ultimately, the platform choice should be driven by the quality of data, project needs, and the intended use of the data generated. In a time-and resourceconstrained environment, it is not possible to evaluate all emergent technologies available in the market; we hope that this review gives the reader some of the information needed to decide which technology he/ she may want to consider evaluating to support their drug development program in comparison to the options they already have in their hands.