Effect of Trastuzumab–HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications. Modifications in the CDRs of trastuzumab were thus compared between the free antibody and the trastuzumab–HER2 complex when stressed under physiological conditions at 37°C. Complex formation and stability of the complex upon stressing were assessed by size-exclusion chromatography. Deamidation of light-chain Asn-30 (Lc-Asn-30) was extensive when trastuzumab was stressed free but reduced about 10-fold when the antibody was stressed in complex with HER2. Almost no deamidation of heavy-chain (Hc-Asn-55) was detected in the trastuzumab–HER2 complex, while deamidation was observed when the antibody was stressed alone. Hc-Asp-102 isomerization, a modification that critically affects biological activity, was observed to a moderate degree when the free antibody was stressed but was not detected at all in the trastuzumab–HER2 complex. This shows that complex formation has a major influence on critical modifications in the CDRs of trastuzumab.

Spontaneous Isomerization of Asp387 in Tau is Diagnostic for Alzheimer's Disease: An Endogenous Indicator of Reduced Autophagic Flux

Amino acid isomerization is a spontaneous chemical modification potentially related to the underlying causes of Alzheimer's disease (AD). We demonstrate that data-independent acquisition mass spectrometry can be used to characterize isomerization in complex protein mixtures. Examination of a large cohort of brain tissue samples revealed a striking relationship between isomerization of tau and AD status. Surprisingly, isomerization was found to be more abundant in both autosomal dominant and sporadic AD samples relative to controls. We hypothesize that lower autophagic flux in AD brains accounts for these results. Additional data, including quantitative analysis of proteins related to autophagy, strongly support this hypothesis. For example, isomerization of tau is positively correlated with levels of p62, a recognized indicator of autophagic inhibition. In sum, the data suggest strong ties between isomerization and autophagic flux, which may therefore represent a promising target for future investigations into the therapy and prevention of AD.

Linoleic acid isomerization ability of Lactobacillus spp. isolated from Vietnamese human intestinal origins

Conjugated linoleic acid (CLA) have been shown to exert numerous health benefits, including anti-carcinogenic, anti-atherogenic, anti-diabetic, antiobesity, cholesterol reducing, antioxidant, anti-microbial, immune system modulator and growth-stimulating properties. In human, CLA is produced from Linoleic acid (LA) by gut bacteria. In this study, nineteen Lactobacillus (Lac.) strains isolated from human feces were studied to determine their ability to metabolize LA. The bacteria were grown in the liquid form of anaerobic MRS medium supplemented with 0.5 mg/mL LA. The linoleate isomerase activity in bacteria grown on MRS medium was determined by Gas chromatograpy. The results indicated that 4 out of 19 strains, including strains Lac.02, Lac.05, Lac.14 and Lac.16 are capable of producing about 40-50 μg/mL CLA from LA. Among them, the highest ability to produce CLA from LA is Lac.02 strain. In the production of CLA from LA, enzymes involved in this metabolism in Lactobacillus act as catalysts of hydration/dehydration (CLA-HY), oxidation of hydroxy groups/reduction of oxo groups (CLA-DH), migration of carbon-carbon double bonds (CLA-DC), and saturation of carbon-carbon double bonds (CLA-ER). The cla-dh, cla-dc, cla-hy and cla-er genes that encode enzymes CLA-DH, CLA-DC, and CLA-ER had been found in all Lac.02, Lac.05, Lac.14 and Lac.16 strains. Gas chromatography traces indicated that these strains produced the same compounds, which was subsequently identified as cis-9, trans-11, and trans-10, cis-12 CLA. In the next study, we will optimize the conditions such as substrate concentrations, pH values, temperature and culture time of each strain to obtain the best rerults.

An Omalizumab Biobetter Antibody With Improved Stability and Efficacy for the Treatment of Allergic Diseases

The critical role of IgE in allergic diseases is well-documented and clinically proven. Omalizumab, a humanized anti-IgE antibody, was the first approved antibody for the treatment of allergic diseases. Nevertheless, omalizumab still has some limitations, such as product instability and dosage restriction in clinical application. In this study, we attempted to develop an omalizumab biobetter antibody with the potential to overcome its limitations. We removed two aspartic acid isomerization hotspots in CDRs of omalizumab to improve antibody candidate’s stability. Meanwhile, several murine amino acids in the framework region of omalizumab were replaced with human source to reduce the potential immunogenicity. Yeast display technology was then applied to screen antibody candidates with high binding affinity to IgE. Moreover, YTE mutation in Fc fragment was introduced into the candidates for extending their serum half-life. A lead candidate, AB1904Am15, was screened out, which showed desired biophysical properties and improved stability, high binding affinity and elevated potency in vitro, prolonged half-life in human FcRn transgenic mouse, and enhanced in vivo efficacy in cynomolgus monkey asthma model. Overall, our study developed a biobetter antibody of omalizumab, AB1904Am15, which has the potential to show improved clinical benefit in the treatment of allergic diseases.

Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.