Expanding the Boundaries of Biotherapeutics with Bispecific Antibodies

Abstract

Bispecific antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra^® and Blincyto^®) and many more in clinical trials. The success of bispecific antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecific antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecific antibodies for crossing the blood-brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specific delivery or activity of antibodies. The different molecular solutions to the initial hurdles that limited the development of bispecific antibodies have led to the current diverse set of bispecific or multispecific antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecific antibodies come at the price of additional challenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specificity complicates the selection of relevant species for safety assessment.

Fig. 1

Selected IgG-like bispecific antibody formats. ‘κλ bodies’ (Novimmune) contain a common heavy chain (HC) and employ the difference in light chain (LC) backbones for purifying the bispecific antibody from contaminant products. The ‘common LC’ format scheme represents the format used by Regeneron; the red star symbolizes the star substitution in one of the heavy chains. In the ‘knob-into-hole’ format (Genentech), the three mutations creating the ‘hole’ and the single mutation creating the ‘knob’ are indicated. In the ‘charge pair’ antibody format (Amgen), the mutations within the C_H3 domain that favor heterodimeric HC association are indicated. The ‘CrossMAb’ (Roche) format employs the knob-into-hole approach for correct HC pairing, as well as a domain swap to enable orthogonal LC–HC pairing. The scheme depicts a CrossMAb where the C_L and C_H1 domains have been swapped

Fig. 2

Selected fragment-based bispecific antibody formats. The ‘BiTE’ (bispecific T-cell engager) format (Amgen) consists of two scFvs connected with a Gly/Ser peptide linker. DARTS (Dual Affinity Re-Targeting proteins, MacroGenics) are diabodies containing an inter-Fv disulfide for increased stability that results in a structure that is rigid and compact. ‘TandAbs’ (Affimed) are dimers of scFvs containing the V_HA/V_LB/V_HB/V_LA domain organization where short linkers favor the correct assembly of the Fvs. The resulting molecule is bivalent for each specificity. Single domain antibodies like V_HH and shark single variable new antigen receptor domain antibody fragments (VNARs) can be easily fused to create bispecific ‘nanobodies’ and ‘VNARs’

Fig. 3

Selected appended-IgG bispecific antibody formats. Shown are the symmetric fusion of Fv fragments to generate ‘DVD-IgGs’ (AbbVie) and the asymmetric fusion of an Fv domain to create an ‘V_H/V_L-IgG’ [238], bivalent for the mAb specificity and monovalent for the specificity conferred by the Fv. The symmetric fusion of scFvs to the C-termini of the HCs generate a ‘scFv-IgG’ (MedImmune), a bivalent molecule for each binding specificity. The represented ‘Fab-IgG’ contains symmetric fusion of Fab fragments in the N-termini of the HCs. Indicated are charge mutation pairs required to direct the cognate association of the heavy and light chains

Fig. 4

Mechanisms of action enabled by bispecific antibodies. (A) Redirecting effector cells for cytotoxicity. The bispecific antibody is designed to simultaneously engage a cancer and effector cell (i.e., T cells) resulting in effector cell activation and cancer cell death. (B) Simultaneous blockade of two pathways. Targeting of two receptors on cancer cells (i.e., EGFR and MET) can result in a more potent inhibition of cell growth. (C) Transcytosis across the blood–brain barrier (BBB). One arm of the bispecific antibody recognizes a receptor that promotes shuttling across the BBB (e.g., transferrin receptor) and the other targets a pathway in the brain involved in neuropathology. (D) Hyperclustering of receptors for internalization. Bispecific antibodies can induce hyperclustering of receptors and antibody internalization, which can be exploited to increase delivery of antibody–drug conjugates. (E) Forced interaction of membrane or membrane-associated proteins. Bispecific antibodies can be used to mimic factors involved in forming productive membrane protein complexes (i.e., factor VIII to enable clotting). (F) Tissue-specific delivery. Targeted delivery of bispecific antibodies to tissues can reduce liabilities from systemic administration (i.e., targeting TNF on macrophages)