Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies.

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Use of Multifactorial Treatments to Address the Challenge of Translating Experimental Myocardial Infarct Reduction Strategies.

Myocardial tissue damage that occurs during an ischemic event leads to a spiraling deterioration of cardiac muscle structural and functional integrity. Reperfusion is the only known efficacious strategy and is the most commonly used treatment to reduce injury and prevent remodeling. However, timing is critical, and the procedure is not always feasible for a variety of reasons. The complex molecular basis for cardioprotection has been studied for decades but formulation of a viable therapeutic that can significantly attenuate myocardial injury remains elusive. In this review, we address barriers to the development of a fruitful approach that will substantially improve the prognosis of those suffering from this widespread and largely unmitigated disease. Furthermore, we proffer that ephrinA1, a candidate molecule that satisfies many of the important criteria discussed, possesses robust potential to overcome these hurdles and thus offers protection that surpasses the limitations currently observed.

Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs.

Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.

Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12.

2G12 is one of only a few cloned antibodies with broadly neutralizing specificity to HIV-1 envelope proteins. Crystallographic and electron microscopic (EM) data showed that the Fab arms are locked together via a novel VH domain exchange. Both the conventional and the unprecedented additional VH-VH antigen binding sites show specificity for high mannose oligosaccharides on the silent face of gp120. We have now extended the EM and biochemical analysis of 2G12. Unligated 2G12 IgG1 molecules clearly show paired (parallel attached) Fab arms in the "doughnut" configuration attached to the Fc both in individual and computationally averaged images. A minority of the IgG molecules in the 2G12 prep showed the open "Y" configuration of conventional IgG. The averaged EM image compares well to the atomic structure model of 2G12. Papain digests of 2G12 yielded paired Fab arms (Fab dimer), as observed by EM, which dissociated into Fab-sized fragments in non-reducing SDS-PAGE. Purified 2G12 reduced and alkylated H and L chains can reassociate to form IgG molecules with the Fab dimer configuration and can combine with L and H chains from conventional human IgG to form hybrid molecules. 2G12 is heavily aggregated following brief acid exposure possibly as a result of its unique structure. A model of the aggregation process is proposed. An anti-Id MAb was shown by EM to react with neither the conventional nor additional antigen binding sites, but bound to the lateral faces of the Fab arms of intact, reduced and alkylated, and reconstructed 2G12 molecules. Efforts to identify IgG molecules with a similar intertwined Fab dimer structure in a large IgG pool were unsuccessful.

Modulation of antigenicity related to changes in antibody flexibility upon lyophilization.

Lyophilization is frequently used to increase the shelf-life of biopharmaceuticals containing antibodies. A case in which an anti-idiotypic antibody, MMA 383, substantially lost its in vivo immunogenic properties although the protein was not degraded, is investigated. The scanning transmission electron microscope allowed the MMA 383 Fab and Fc moieties to be resolved. By averaging the single antibodies, the angle between the Fab moieties can be calculated. Non-lyophilized antibodies displayed a wider range of shapes than their reconstituted, lyophilized counterparts. Accordingly, the angle between the two Fab fragments varied more, indicating greater flexibility. The tryptophan steady-state fluorescence intensity, steady-state fluorescence anisotropy and fluorescence lifetime, were smaller for the lyophilized antibodies. These were also more resistant towards thermal denaturation/aggregation. Circular dichroism spectra detected temperature-dependent differences between the two antibody types in the 236 nm region. The subtle but reproducible structural changes induced by lyophilization may be related to the loss of in vivo immunogenic properties.

Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.

The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound Fab fragment and IgG structures suggested that conformational changes occur about elbow axes upon bivalent attachment as was previously predicted. No significant density was observed for the Fc fragment, which adds further evidence for a high degree of mobility about the hinge region.

Proton nuclear magnetic resonance studies of the structure of the Fc fragment of human immunoglobulin G1: comparisons of native and recombinant proteins.

The structure of the Fc fragment of human IgG1 immunoglobulin is compared for the native and recombinant proteins. A recombinant human Fc fragment was expressed by an E. coli system [Kitai K., Kudo T., Nakamura S., Masegi T., Ichikawa Y. and Horikoshi K. (1988) Appl. Microbiol. Biotechnol. 28, 52-56]. The recombinant protein, which presumably lacks oligosaccharides, was used along with the native human Fc fragment obtained by proteolytic digestion of a myeloma IgG1 protein. 1H NMR has been employed along with circular dichroism and fluorescence spectroscopy to discuss the structure of these two types of proteins. It has been concluded that (1) the overall structure of the recombinant protein is quite similar to that of the native protein, which possesses asparagine-linked oligosaccharides, but (2) a significant difference in structure exists in the neighborhood of the glycosylation site. The difference in the effector functions for the two kinds of the Fc proteins has been briefly discussed in terms of the structural change detected by 1H NMR.