Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display

Krah, Simon; Schröter, Christian; Eller, Carla; Rhiel, Laura; Rasche, Nicolas; Beck, Jan; Sellmann, Carolin; Günther, Ralf; Hock, Björn; Kolmar, Harald; Becker, Stefan

doi:10.1093/protein/gzw077

Cited by 31 publications

(53 citation statements)

References 42 publications

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“…Reconstituted CEACAM5xCEACAM6 (C5xC6) bsAb showed a sub-nanomolar K D value (0.3 nM) for binding soluble recombinant CEACAM5 and 0.4 nM for binding soluble recombinant CEACAM6, which was comparable to the K D values of reference bsAb C5xC6, as previously described (Figure 4a,b). [33] Simultaneous binding of reconstituted C5xC6 to both antigens via stepwise association was retained after PTS compared to reference C5xC6 bsAb ( Figure 4c). In congruence, kinetic parameters of several additional reconstituted bsAb were comparable to their genetically fused references, as depicted in Table 1.…”

Section: Characterization Of Reconstituted Antibodiesmentioning

confidence: 95%

Intein mediated high throughput screening for bispecific antibodies

Hofmann

Schmidt

Ciesielski

et al. 2020

mAbs

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Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.

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Section: Characterization Of Reconstituted Antibodiesmentioning

confidence: 95%

Intein mediated high throughput screening for bispecific antibodies

Hofmann

Schmidt

Ciesielski

et al. 2020

mAbs

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“…Upon yeast mating diploid cells are generated where a surface-anchored heavy chain assembles with the secreted light chain to form a Fab fragment (Fig. 1 ) [ 23 , 28 ]. Library diversities were calculated to be approximately 2 × 10 9 unique clones for each of the RTK VH and Vk libraries.…”

Section: Resultsmentioning

confidence: 99%

“…All used plasmids, yeast strains and media have been described elsewhere [ 23 , 24 ]. In brief, modified pYD1 yeast surface display vectors were used (Yeast Display Vector Kit, version D, #V835-01, Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…Isolated pYD plasmids of selected clones were used for subcloning of VH/VL regions into pTT5 vectors using the Expresso CMV-based system (Lucigen cloning) to allow full-length IgG expression in mammalian cells as described elsewhere [ 23 ]. In brief, cloned constructs were expressed in Expi293 cells after Expifectamin-mediated transient transfection according to the manufacturer´s instructions (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

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Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

et al. 2018

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In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species’ cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.Electronic supplementary materialThe online version of this article (10.1007/s12033-018-0109-0) contains supplementary material, which is available to authorized users.

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“…To avoid promiscuous association of light chains to heavy chains in bispecific antibody production, different antibodies (with different ligand binding specificity) using one common light chain have been selected from phage or yeast display libraries, providing a popular solution to the light chain pairing problem [50][51][52] . However, this restriction on light chain reduces the sequence space that can be explored for binding affinity/specificity and downstream developability.…”

Section: Introductionmentioning

confidence: 99%

LegoBody: facile generation of bispecific and multi-specific antibodies

Lang

Yang

Bidlingmaier

et al. 2019

Preprint

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Bispecific and multi-specific antibodies are capable of recognizing multiple ligands simultaneously or synergistically, creating complex biological interactions not achievable by monoclonal antibodies, thus expanding opportunities for novel therapy development.With the large number of monoclonal antibodies either approved or under clinical development, there are numerous opportunities to combine their specificities to further improve therapeutic potential. Although simple in concept, clinical development of biand multi-specific antibodies face several challenges, chief of which is how to efficiently and reliably produce bispecific and multi-specific antibodies with expected specificity and desired biophysical properties. In this study, we developed a modular approach that uses temporary linkers to enforce proper chain pairing and proteases such as thrombin to remove those linkers from the final product. Combined with the 'knob-into-hole' design, we can generate IgG-like, bi-or multi-specific antibodies from any pre-existing monoclonal antibodies. The approach is highly versatile and applicable to any monoclonal antibody pair or panel, expediting evaluation and therapeutic development of bi-and multi-specific antibodies.

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Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display

Cited by 31 publications

References 42 publications

Intein mediated high throughput screening for bispecific antibodies

Intein mediated high throughput screening for bispecific antibodies

Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

LegoBody: facile generation of bispecific and multi-specific antibodies

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