1.2.4. MoA Type 4—"Homing" BsAbs

For the purposes of this review, "homing" BsAbs are molecules in which one arm serves to deliver the molecule to a specific, often hard-to-reach location. There are multiple examples with a diverse range of therapeutic targets. Several BsAbs have been developed that are able to cross the blood-brain barrier by targeting the transferase receptor [63–65]. Once across the barrier, the non-transferase receptor arm can target the therapeutic target of interest (typically amyloid beta-protein and other Alzheimer's Disease targets). Additionally, there is an example of a tandem scFv BsAb that targets activated platelets and sca-1, helping to bring stem cells to the location of injury; it is being explored for the treatment of myocardial infarction [66]. An aCD63/aHER2-ADC has been developed, in which binding to CD64 targets the molecule to the lysosome while the aHer2 portion provides tumor specificity, leading to a more efficient release of the conjugated drug [67]. There is also an example of a BsAb with one epitope designed to gain entry into the late endosome, where it is able to neutralize Ebola virus [68].

#### *1.3. Challenges and Opportunities of BsAb Bioassay Development*

Concurrent to the development of these complex biological products with multiple modalities is the need to develop bioassays that are not only accurate and reproducible, but also adequately reflective of the proposed mechanism(s) of action. Well-developed bioassays are critical to the characterization and control of biological products, as well as to the interpretation of clinical study results. BsAb bioassay development presents a unique set of challenges for assay design, such as the ability to fulfill the desired performance of the assay (i.e., to capture the dual activities and potential synergistic effects of the molecule) preferably using a single assay format, and to detect multifaceted structural changes [69]. Depending on the molecule's MoA, several bioassays might be necessary for characterization in addition to a main potency assay in the control system. For example, cellkilling, cytokine secretion, receptor internalization [70], effector function (ADCC, ADCP), and surface marker expression assays might need to be developed for the characterization of bispecific molecules for later stages of product development in addition to the one most MoA-relevant bioassay selected and validated for release, stability, and comparability testing for product licensure. A number of technologies were developed to overcome these challenges to characterize BsAb, and selected case studies are described in the Section 3.

#### **2. Strategies and Considerations for BsAb Bioassay Development**
