Russell body phenotype is preferentially induced by IgG mAb clones with high intrinsic condensation propensity: Relations between the biosynthetic events in the ER and solution behaviors in vitro

Abstract

The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER.

Overexpression of cryoglobulin‐like single‐chain antibody induces morular cell phenotype via liquid–liquid phase separation in the secretory pathway organelles

“…The biosynthetic steps of such cryoglobulins have also been rarely investigated because of the difficulty in isolating patient‐derived plasma cell clones producing such cryoglobulins. Furthermore, although Russell body and related cellular phenotypes of immunoglobulin‐expressing cells have been known for more than a century, the precise reasons why such phenotypes arise and why different types of cell phenotypes come about remain enigmatic .…”

“…Recent studies have shown that intracellular phase separation events in the endoplasmic reticulum (ER) contribute to the induction of certain classes of cell‐ular phenotypes in immunoglobulin‐expressing cells . Depending on the intrinsic physicochemical properties of a cargo (e.g.…”

“…When a given IgG clone possesses such propensities, the IgG protein often exhibits similar phase separation behaviors in test tubes at high concentrations or in the kidney glomeruli in vivo . Similarly, if IgG clones have different types of condensation propensities such as gelation, particulate formation, amorphous aggregation or high viscosity, these immunoglobulins tend to induce Russell body phenotypes during protein overexpression at a steady state and under ER‐to‐Golgi transport block . When it comes to morular cell phenotypes, it is still unknown what types of physicochemical properties embedded in the overexpressed secretory cargo would contribute to the generation of such cell morphology.…”

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

“…Historically, a number of computational and analytical screening methods have been used to predict aggregation [28][29][30] and high concentration formulation risks. 17,[31][32][33][34][35][36] While these approaches can provide valuable information and help screening of potential lead candidates during discovery and early stage development, they do not necessarily provide insight into the underlying molecular mechanisms involved. With regard to high concentration challenges, the availability of methodologies that can probe native intermolecular interactions at the structural and molecular level can effectively guide corresponding protein engineering and formulation efforts designed to mitigate the development risks.…”

Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD

“…Similar structures can be found in plasma cells undergoing excessive synthesis of IgGs, especially as a manifestation of physiological abnormalities like multiple myeloma, inflammatory diseases, and autoimmune disorders (Decourt, Galea, Sirac, & Cogné, ; Jiang et al, ). Nevertheless, RBs have also been identified in recombinant IgG producing cell lines such as CHO and HEK293 (Hasegawa et al, ; Hasegawa, Woods, Kinderman, He, & Lim, ; Stoops, Byrd, & Hasegawa, ) as well as during overexpression of bispecific antibody formats in CHO cells (Mathias et al, ). Interestingly, this vesicularized phenotypic appearance of the ER has also been described independently in the context of several different and very diverse conditions including oxidative stress (Hendershot et al, ; Wei et al, ), downregulation of chaperones (Lee, Chu, Iwakoshi, & Glimcher, ; Li et al, ), and parts of the ERAD machinery (Sun et al, ).…”