Influence of Isoelectric Points (pI) to the Permeability of Single Domain Antibody (sdAb)
The positive charges present on the surface of sdAbs lead to high isoelectric points (pI), which may play a critical role in brain penetration of these sdAbs across the BBB. Along with over a decade of extensive experience and advanced Super™ intrabody or transbody development platforms, Creative Biolabs is a well-recognized global leader and professional in supporting a broad range of transbody and intrabody development projects to satisfy various specific demands.
Isoelectric Points (pI) of sdAb
The theoretical isoelectric points of sdAb are observed covering a wide range (pI values between 5 and 10), which portends different degrees of physical and/or biological stability between the sdAbs. Due to the small size of these sdAb molecules ranging from 13-16 kDa, their isoelectric point is mainly determined by the different amino acid compositions of the complementary determining region (CDR) regions. This might be exploited in the development of different therapeutic or imaging agents. It is important to note that, in general, an extremely high or low isoelectric point will render some of the sdAbs unsuitable for in vivo use. As previous research demonstrated in some sdAbs, decreasing the isoelectric point not only enhances the solubility but also results in improved levels of production.
The Influence of pI to Permeability of sdAb
The factors influencing sdAbs transit across the blood-brain barrier (BBB) are poorly understood. It is demonstrated that the small molecular size of sdAbs is an essential parameter for their brain penetration compared to some large antibody fragments. Furthermore, the basic pI also has appeared to be an important parameter to influence the permeability of sdAb. The sdAb expression products with a highly positively charged pI (e.g., pI=9.4) have been found to enhance permeability and to cross the BBB spontaneously. Such sdAbs not only gain access to the brain but are even found to penetrate cells and bind to intracellular proteins. These sdAbs readily transmigrate across the BBB in vivo after peripheral injection, without the need for any invasive or hazardous procedures (such as the opening of the barrier using chemical or ultrasound sonication). This indicates that the BBB crossing of sdAb is not energy-dependent, suggesting active transport is not involved. The transmigration of basic sdAbs through the BBB probably occurs by adsorptive-mediated endocytosis initiated by interactions with the negatively charged luminal surface of the BBB, which contributes to the transcytosis into the brain parenchyma. This transcytosis mechanism has been earlier identified to shuttle basic proteins and peptides across the BBB.
Fig. 1 Single domain antibody (sdAb) crossing the blood-brain barrier (BBB).1
The basicity of sdAbs can be increased by exchanging the carboxyl groups of the sdAbs with positively charged amino groups, thereby increasing the isoelectric point. These cell-penetrating sdAbs display a set of characteristics not observed from conventional antibodies and derived fragments. Their small size enhances the diffusion in tissues, and the basic pI is linked to the ability of cell-penetrating of the sdAbs. Furthermore, the intracellular binding depends on the hyperstability of sdAbs. Understanding the precise mechanism of cell-penetrating sdAb will be important for optimizing sdAbs as a specific transporter (such as VHH-GFP fusion protein to cross the BBB and to label specific targets in vivo).
Shifting the pI of therapeutic sdAbs to a basic level facilitates the permeability of these sdAbs to cross the BBB. Based on our extensive experience and our Super™ intrabody and transbody development platforms, Creative Biolabs has won a high reputation among our worldwide clients for successfully developing a series of high-quality transbody and intrabody. If you are interested in our services, please send us an inquiry for detailed information.
Reference
- Rissiek, Björn, Friedrich Koch-Nolte, and Tim Magnus. "Nanobodies as modulators of inflammation: potential applications for acute brain injury." Frontiers in cellular neuroscience 8 (2014): 344. Distributed under Open Access license CC BY 4.0, capture the part A of original Figure 1.
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