(a) Illustration of PD-L1 bioassay: Anti-PD-L1 arm of anti-PD-L1/CD40L BsAbFP inhibits the PD-1/PD-L1 pathway leading to the activation of luciferase reporter gene in Jurkat/PD-1/NFAT-Luc cell; (b) Illustration of CD40L bioassay: CD40L arm of anti-PD-L1/CD40L BsAbFP binds to CD40 leading to the activation of luciferase reporter gene in Raji/NFB-Luc cell; (c) Representative PD-L1 bioassay graph comparing dose-dependent response between of anti-PD-L1/CD40L BsAbFP and anti-PD-L1 mAb; (d) Representative CD40L bioassay graph comparing dose-dependent response between anti-PD-L1/CD40L BsAbFP and CD40L. 2.3. increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this three-cell-in-one dual target reporter gene bioassay is suitable for assessing potency, structure-function and crucial quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy. Keywords: Rabbit Polyclonal to RPS12 bioassay, bispecific antibody, potency, mechanism of action, qualification 1. Introduction Immunotherapeutic antibodies have demonstrated superior tolerability and major improvements in long-term survival in cancer patients [1,2]. Use of immune checkpoint inhibitors or costimulatory molecules as Hexaminolevulinate HCl a target for immunotherapy has become one of the promising approaches in the field of malignancy therapy. Among antibody-based cancer therapies, bispecific antibodies (BsAbs), and antibody-fusion proteins (BsAbFPs), that bind to two different antigen targets, are an emerging class of biotherapeutics for cancer patients [3]. Following the regulatory approval of blinatumomab, a number of BsAbs have undergone clinical development. BsAbs can be classified as cytotoxic effector cell redirectors, tumor-targeted immunomodulators and dual immunomodulators. In recent years, numerous BsAbs targeting the costimulatory (e.g., OX40, CD40, 4-1BB etc.), coinhibitory pathways (PD-1, CTLA-4, TIM-3, TIGIT etc.) or tumor antigens (MUC1, MUC16) have been developed and are in preclinical and clinical investigations. The BsAb format is often a combination of two distinct variable regions derived from two different parental monospecific antibodies. In other cases, such as that presented here, both Fab arms bind the same target, and an additional target-specificity is usually added via protein fusion around the C-terminus of the Fc domain name. Unlike binding to single-antigen on target cells, BsAbs possess the ability to bind to two antigens simultaneously. The ability of BsAbs to simultaneously bind two different antigens enables a variety of potential mechanism of actions (MOAs), such as an improved cytotoxic potential by bridging cells in-trans, synergistic effects, and receptor crosslinking leading to enhanced inhibition or degradation of target proteins from the cell surface with higher binding specificity and tumor selectivity [4]. Programmed death 1 (PD-1) is usually expressed on activated T cells and has emerged as an important mediator for negatively regulating T cell responses. It acts as a key checkpoint molecule in tumor-induced immune suppression [5]. Antibodies targeting PD-1/PD-L1 checkpoint stimulate the immune system to keep the tumor in check by promoting the T cells response from attenuating effects of PD-1. When engaged by one of its ligand PD-L1 or PD-L2, PD-1 inhibits the kinases that are involved in T cell activation and downstream signaling pathways that further suppress the effector function including exhaustion of T cell immune response [6,7]. Blockade of the conversation of PD-1 with PD-L1 or PD-L2 has been shown to enhance the antitumor activity of T cells [7,8]. CD40 Hexaminolevulinate HCl is usually a membrane protein belonging to TNF receptor (TNFR) superfamily that functions as a key costimulatory molecule for activating both innate and adaptive immune system [9]. It also has a non-redundant role in antibody class-switching [10]. It is primarily expressed on the surface of B cells, Hexaminolevulinate HCl dendritic cells, macrophages and on most of their neoplastic counterparts [11]. CD40 binding to its ligand (CD40L) exerts profound effects on these.