Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation

Fujii, Tomohiro; Ito, Kenichiro; Takahashi, Kazutoshi; Aoki, Tsubasa; Takasugi, Rika; Seki, Takuya; Iwai, Yusuke; Watanabe, Tomohiro; Hirama, Ryusuke; Tsumura, Ryo; Fuchigami, Hirobumi; Yasunaga, Masahiro; Matsuda, Yutaka

doi:10.1021/acsmedchemlett.3c00414

Cited by 3 publications

(2 citation statements)

References 38 publications

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“…The need for scalable and robust methods to screen bispecific antibody constructs was recently addressed in the “AJICAP second generation” strategy, which employs affinity peptides pre-orientating amine reactive groups to attach Fab fragments to antibodies. 38 However, this is a two-step synthesis which requires to eliminate excess reaction compounds in between. Alternative approaches for generating dual binding constructs include intein-based conjugation 39 , the SypTag/SypCatcher approach 40 , SynAbs strategy 41 or controlled Fab arm exchange 42 .…”

Section: Resultsmentioning

confidence: 99%

Welding PROxAb Shuttles: A modular approach for generating bispecific antibodies via site-specific protein-protein conjugation

Lehmann,

Schneider,

Tonillo

et al. 2024

Preprint

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Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), it has shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically in cells expressing the antibodies target. The chemoenzymatic approach described here provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications but could also be used to combine antibody and VHH binders to generate bispecific antibodies for further applications.

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Section: Resultsmentioning

confidence: 99%

Welding PROxAb Shuttles: A modular approach for generating bispecific antibodies via site-specific protein-protein conjugation

Lehmann,

Schneider,

Tonillo

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The combination of VHHs of different target specificities with antibodies of additional specificities would allow to quickly generate combinatorial libraries of bispecific antibodies to screen for optimal target combinations. The need for scalable and robust methods to screen bispecific antibody constructs was recently addressed in the “AJICAP second generation” strategy, which employs affinity peptides preorientating amine reactive groups to attach Fab fragments to antibodies . However, this is a two-step synthesis that requires eliminating excess reaction compounds in between.…”

Section: Resultsmentioning

confidence: 99%

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein–Protein Conjugation

Lehmann,

Schneider,

Tonillo

et al. 2024

Bioconjugate Chem.

View full text Add to dashboard Cite

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

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Chemical Production of Cytotoxic Bispecific Antibodies Using the Ugi Multicomponent Reaction

Vaur,

Koutsopetras,

Erb

et al. 2024

ChemBioChem

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Bispecific antibodies (bsAbs) have recently emerged as a promising platform for the treatment of several conditions, most importantly cancer. Based on the combination of two different antigen‐binding motifs in a single macromolecule; bsAbs can either display the combined characteristics of their parent antibodies, or new therapeutic features, inaccessible by the sole combination of two distinct antibodies. While bsAbs are traditionally produced by molecular biology techniques, the chemical development of bsAbs holds great promises and strategies have just begun to surface. In this context, we took advantage of a chemical strategy based on the use of the Ugi reaction for the site‐selective conjugation of whole antibodies and coupled the resulting conjugates in a bioorthogonal manner with Fab fragments, derived from various antibodies. We thus managed to produce five different bsAbs with 2:1 valency, with yields ranging from 20% to 48%, and showed that the affinity of the parent antibody was preserved in all bsAbs. We further demonstrated the interest of our strategy by producing two other bsAbs behaving as cytotoxic T cell engagers with IC50 values in the picomolar range in vitro.

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Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation

Cited by 3 publications

References 38 publications

Welding PROxAb Shuttles: A modular approach for generating bispecific antibodies via site-specific protein-protein conjugation

Welding PROxAb Shuttles: A modular approach for generating bispecific antibodies via site-specific protein-protein conjugation

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein–Protein Conjugation

Chemical Production of Cytotoxic Bispecific Antibodies Using the Ugi Multicomponent Reaction

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