A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

A Comprehensive Analysis of Novel Disulfide Bond Introduction Site into the Constant Domain of Human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bonds can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the H: F130C-L: Q124C, H: F174C-L: S176C, H: V177C-L: Q160C, H: F174C-L: S162C, H: F130C-L: S121C, and H: A145C-L: F116C mutants mostly formed intermolecular disulfide bonds. All these mutants showed increased thermal stabilities compared to those without intermolecular disulfide bonds. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C-F118C mutant showed only a slight decrease in binding activity and β-helix content, owing to adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals the introduction of intermolecular disulfide bonds in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Rapid and robust antibody Fab fragment crystallization utilizing edge-to-edge beta-sheet packing

Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fab structures, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call “Crystal Kappa”, enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.

Novel bioanalytical method for the characterization of the immune response directed against a bispecific F(ab) fragment

Aim: The work was aimed at developing a bioanalytical approach to identify immunogenic parts of a bispecific F(ab) fragment and to characterize the immune response seen in a preclinical study. Experimental: The bioanalytical method consists of a set of domain detection assays that use germlined variants of the drug. Results: The method demonstrated that anti-drug antibodies (ADAs) were predominantly directed against both antigen-binding sites of the drug. Conclusion: The method was capable to discriminate between ADAs directed against one of the antigen-binding sites, both sites or the constant domain, allowing for an estimation of the relative binding prevalence for these subunits. The developed approach provides a practical and robust solution for exploratory characterization of ADAs against multidomain biotherapeutics.

First-order justification logic with constant domain semantics

Justification logic is a term used to identify a relatively new family of modal-like logics. There is an established literature about propositional justification logic, but incursions on the first-order case are scarce. In this paper we present a constant domain semantics for the first-order logic of proofs with the Barcan Formula (FOLPb); then we prove Soundness and Completeness Theorems. A monotonic semantics for a version of this logic without the Barcan Formula is already in the literature, but constant domains require substantial new machinery, which may prove useful in other contexts as well. Although we work mainly with one system, we also indicate how to generalize these results for the quantified version of JT45, the justification counterpart of the modal logic S5. We believe our methods are more generally applicable, but initially examining specific cases should make the work easier to follow.

CUT AND GAMMA I: PROPOSITIONAL AND CONSTANT DOMAIN R

The main object of this article is to give two novel proofs of the admissibility of Ackermann’s rule (γ) for the propositional relevant logic R. The results are established as corollaries of cut elimination for systems of tableaux for R. Cut elimination, in turn, is established both nonconstructively (as a corollary of completeness) and constructively (using Gentzen-like methods). The extensibility of the techniques is demonstrated by showing that (γ) is admissible for RQ* (R with constant domain quantifiers). The status of the admissibility of (γ) for RQ* was, to the best of the author’s knowledge, an open problem. Further extensions of these results will be explored in the sequel(s).