“…1a , but also with a variety of additional format derivatives, as long as they harbor Fc regions that can be paired with exchange-driving dummies. This includes Fabs, sterically constrained Fab-arms (e.g., Contorsbodies) or other binders such as single-chains, single domains or scaffold binders 1 – 3 , 7 , 8 , 33 , 34 . Figure 1b shows the combination of educts with binding entities in various formats, such as entities with Fab arms attached to the Fc N-terminal, C-terminal, or both.…”
Section: Resultsmentioning
confidence: 99%
“…Coordinates for those structures are deposited as PDB 6YT7 (with knob-dummy) and 6YSC (hole dummy), details of the structure determinations are provided in “Methods” and Supplementary Table 1 . Figure 2e, f shows molecules with perfect interface complementarity of bsAb Fc (disulfide-stabilized KiH Fc with original salt bridge), and of the dummy-dimer (KiH without interchain disulfide and perfect inverted salt bridge) resulting from the exchange reaction (PDB 5HY9 for the previously published KiH Cys–Cys 34 , and PDB 6YTB for the dummy-dimer deposited as part of this work). Fig.…”
Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.
“…1a , but also with a variety of additional format derivatives, as long as they harbor Fc regions that can be paired with exchange-driving dummies. This includes Fabs, sterically constrained Fab-arms (e.g., Contorsbodies) or other binders such as single-chains, single domains or scaffold binders 1 – 3 , 7 , 8 , 33 , 34 . Figure 1b shows the combination of educts with binding entities in various formats, such as entities with Fab arms attached to the Fc N-terminal, C-terminal, or both.…”
Section: Resultsmentioning
confidence: 99%
“…Coordinates for those structures are deposited as PDB 6YT7 (with knob-dummy) and 6YSC (hole dummy), details of the structure determinations are provided in “Methods” and Supplementary Table 1 . Figure 2e, f shows molecules with perfect interface complementarity of bsAb Fc (disulfide-stabilized KiH Fc with original salt bridge), and of the dummy-dimer (KiH without interchain disulfide and perfect inverted salt bridge) resulting from the exchange reaction (PDB 5HY9 for the previously published KiH Cys–Cys 34 , and PDB 6YTB for the dummy-dimer deposited as part of this work). Fig.…”
Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.
“…Many fragment-based BsAbs are made by combining scFv fragments of different specificities (see Figure 1g-l), and they often self assemble from a single polypeptide chain (no opportunity for chain mispairing) [17]. Their small size can lead to better tissue penetration, and it has been postulated that their small size and conformational flexibility enable a more potent receptor activation, for example when bridging two cell types, compared to their larger counterparts [7,10]. However, they tend to have very short serum half lives due to the lack of an Fc domain.…”
Section: Diverse Formats Of Bsabmentioning
confidence: 99%
“…BsAbs are typically designed to possess the epitope specificity and manufacturability of a conventional monoclonal antibody (mAb) but are engineered to bind two distinct targets instead of one. The actual structure of a BsAb can vary widely, and depends on a number of factors including the intended mechanism of action (MoA) of the BsAb and desired pharmacokinetic/pharmacodynamic (PK/PD) properties [7,8]. Development and commercialization of BsAbs, to engage multiple targets using only one therapeutic, has gained significant attention recently, shifting industry focus and investments on this effective therapeutic strategy.…”
Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules.
“…Many biotherapeutics currently in pre-clinical research and early development are complex molecules, mostly bispecific antibodies, and require both a specific spatial arrangement and flexibility of their binding moieties to not only specifically bind but also fully elicit their functionality of often several desired MoAs [85,86]. Therefore, in-format screening of bispecific antibodies is desirable and can significantly impact the identification of the best binders' combination [87].…”
Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.
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