Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity.

Aspartic acid (Asp) isomerization is a spontaneous non-enzymatic post-translation modification causing a change in the structure of the protein backbone, which is commonly observed in therapeutic antibodies during manufacturing and storage. The Asps in Asp-Gly (DG), Asp-Ser (DS), and Asp-Thr (DT) motifs in the structurally flexible regions, such as complementarity-determining regions (CDRs) in antibodies, are often found to have high rate of isomerization, and they are considered "hot spots" in antibodies. In contrast, the Asp-His (DH) motif is usually considered a silent spot with low isomerization propensity. However, in monoclonal antibody mAb-a, the isomerization rate of an Asp residue, Asp55, in the aspartic acid-histidine-lysine (DHK) motif present in CDRH2 was found to be unexpectedly high. By determining the conformation of DHK motif in the crystal structure of mAb-a, we found that the Cgamma of the Asp side chain carbonyl group and the back bone amide nitrogen of successor His were in proximal contact, which facilitates the formation of succinimide intermediate, and the +2 Lys played an important role in stabilizing such conformation. The contributing roles of the His and Lys residues in DHK motif were also verified using a series of synthetic peptides. This study identified a novel Asp isomerization hot spot, DHK, and the structural-based molecular mechanism was revealed. When 20% Asp55 isomerization in this DHK motif occurred in mAb-a, antigen binding activity reduced to 54%, but the pharmacokinetics in rat was not affected significantly. Although Asp isomerization of DHK motif in CDR does not appear to have a negative impact on PK, DHK motifs in the CDRs of antibody therapeutics should be removed, considering the high propensity of isomerization and impact on antibody activity and stability.

EP1 prostanoid receptor coupling to G i/o up-regulates the expression of hypoxia-inducible factor-1 alpha through activation of a phosphoinositide-3 kinase signaling pathway.

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE(2)). It is in the family of G-protein-coupled receptors and is known to activate Ca(2+) signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE(2) stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), which can be completely blocked by pertussis toxin, indicating coupling to G(i/o). This up-regulation of HIF-1 alpha occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1 alpha, which involves decreased protein degradation, the up-regulation of HIF-1 alpha by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1 alpha observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1 alpha is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE(2) biosynthesis could up-regulate the expression of HIF-1 alpha and promote tumorigenesis.