Correction: Rapid and robust antibody Fab fragment crystallization utilizing edge-to-edge beta-sheet packing.
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ABSTRACT:
SUBMITTER: PLOS ONE Staff
PROVIDER: S-EPMC8491941 | biostudies-literature | 2021
REPOSITORIES: biostudies-literature
Publications
Correction: Rapid and robust antibody Fab fragment crystallization utilizing edge-to-edge beta-sheet packing.
PloS one 20211005 10
[This corrects the article DOI: 10.1371/journal.pone.0232311.]. ...[more]
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Project description:Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
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Project description:A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.
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Project description:Tetrasulfocyanine (TSC) has been described as a fluorescent probe for tumour imaging. The complex of TSC and the Fab antibody fragment MOR03268 has been crystallized in three different crystal forms. MOR03268 was identified from the HuCAL GOLD library and further optimized to bind TSC with high affinity (Kd = 0.6 nM). For two of the three crystal forms (forms 1 and 2), data sets could be collected to 2.8 and 2.85 A resolution, respectively. Form 1 belongs to space group I222, with unit-cell parameters a = 72, b = 99, c = 154 A. Form 2 belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 77, c = 379 A. Form 3 only diffracted to 8 A and was not analyzed further. Molecular-replacement solutions for forms 1 and 2 were found and rebuilding and refinement is in progress. Form 1 contains one Fab molecule per asymmetric unit, while form 2 harbours two. Judging from the green colour of the crystals, both forms contain the Fab molecule bound to the green TSC dye and in both the hydrolysis-sensitive dye molecule is protected from degradation for several weeks to months. The structures should reveal the molecular basis of the high-affinity recognition of TSC by the Fab molecule MOR03268.
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