The molecule showed greater potency than the combination of monospecific DR4 and DR5 antibodies, suggesting its distinct properties to mediate agonism [38]. molecule with identical domain sequences. Keywords: T cell bispecific, Antibody format, Protein engineering, Geometry, Immune synapse 1.?Background: Different fields, different formats The technical progress in recombinant protein expression and creative antibody engineering brought up many different antibody formats that are reviewed by others in detail [7], [53], [15], [23]. The key message in the literature is that there is no master format that fits all applications. A suited antibody configuration rather UK 356618 depends on the desired biological effect and its underlying structural conditions. Multispecific antibodies are applied for various purposes including (1) receptorCactivation [38], (2) Cblocking [30], (3) Cinternalization [42], (4) Cclustering [8], (5) the association of membrane-associated proteins [26], or (6) the retargeting of cytotoxic effector cells [35] (Fig. 1). Within these application fields, there are several examples that prove that formats influence the bispecific antibody (bsAb) performance. Open in a separate window Fig. 1 Application areas of bispecific antibodies: 1) Receptor activation. 2) Receptor blocking and inhibition of soluble ligand (L) or membrane-bound ligand binding, UK 356618 e.g. of effector cells (E). 3) Receptor internalization. 4) Receptor clustering. 5) Receptor association. 6) Bicellular binding and retargeting of effector cells (E). Receptor activation (1) and induction of downstream signaling to achieve a certain phenotype is the goal of agonistic antibodies. Shi present an IgG-shaped, tetravalent, biparatopic format that contains VH-only binders on each N-terminus. The close distance of paratopes mimics the natural ligand and thereby activates the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR). The distinct antibody geometry was carefully selected and shown to be essential for inducing agonism [52]. The same format was recently published by others who also detected agonism by targeting CD40 [34]. UK 356618 Researchers around Milutinovic have generated a bivalent Y-shaped Surrobody that agonizes the death receptors DR4 and DR5. The molecule showed greater potency than the combination of monospecific DR4 and DR5 antibodies, suggesting its distinct properties to TNFSF13B mediate agonism [38]. Just recently, antibody engineers from Genentech showed agonism via the MerTK (Mer Tyrosine Kinase) receptor pathway on macrophages that led to the phagocytosis of CD20 positive B cells. They made use of a simple 1?+?1 UK 356618 IgG format [25]. Receptor blockade (2) takes currently an important part in cancer immunotherapy approaches. Bispecific, antagonistic molecules that target inhibitory immune checkpoints show promising results in overcoming tumor evasion mechanisms. A prominent example is the dual-blockade of immune checkpoints such as LAG-3 and PD-1, which induces antitumor immunity [30], [61]. The number of ongoing clinical trials with diverse bispecific antibody formats is striking [45], [46], [28]. Whereas, the generation of agonistic antibodies is challenging (they depend on both affinity and intrinsic efficacy), designing UK 356618 blocking antibodies mostly relies on finding high affinity binders that compete with the natural ligand and is therefore seen to be less complex [37]. Antibody-induced receptor internalization (3) plays an essential role in antibody-drug-conjugate (ADC) retargeting [10], [56]. It could be shown that the bivalent binding of EGFR and the thereby triggered dimerization leads to a significantly higher internalization of the receptorCantibody complex than seen with the monovalent format [24], [20]. Niewoehner analyzed different formats to achieve bloodCbrain barrier transcytosis. A monovalent targeting of the transferrin receptor (TfR) lead to a 55-fold higher brain exposure than the bivalent