Monoclonal antibodies targeting by Nanodisc technology

Nanodisc technology offers a means of incorporating integral membrane proteins into a water-soluble, native-like lipid bilayer, making them more suitable for biochemical and biophysical characterization. When a target membrane protein solubilized in detergent is mixed with an optimal phospholipid and membrane scafold protein ratio, nanodiscs are produced (MSP). The removal of the detergent results in the assembly of the target membrane protein into a phospholipid bilayer held together as a helical protein belt by two MSP molecules encircling it. In this type, membrane protein intracellular and extracellular domains are exposed to solvent with hydrophobic transmembrane domains that interact with lipid acyl chains that interact in turn with the MSP amphipathic helices.

It is advisable to establish reagents and methods to assist in the detection of antibodies against them, considering the importance of multi-pass membrane proteins as drug targets and the inherent difficulties in dealing with them.

This research  was publish in nature under title "Nanodisc technology facilitates identifcation of monoclonal antibodies targeting multi-pass membrane proteins"

The author explained "We tried to determine whether nanodisc technology offers a way to determine whether FACS hybridoma cells are specified for targeting integral multi-pass membrane proteins. As a multi-pass integral membrane protein system model, the chimeric VSD4-NavAb ion channel was chosen for our studies".

VSD4-NavAb is a homotetrameric chimeric membrane protein previously described, composed of human Nav1.7 voltage-sensing domain 4 (VSD4) regions grafted onto the bacterial channel NavAb. The protein assembles into a homotetramer in which 6 transmembrane helices are bound by 3 extracellular and 2 intracellular loops and a cytosolic C-terminus is formed by a coiled coil in the monomeric chain. Upon efficient purification and incorporation of VSD4-NavAb into nanodiscs, antigen-specific hybridoma has been shown to be used in FACS. The use of target-loaded nanodiscs for FACS helps us to bypass the need for hybridoma polyclonal plating and greatly enhances the efficacy of antigen-specific hybridoma identification.

For membrane protein structural characterization, display with nanodiscs has previously been shown to be successful and we hypothesised that they will give a novel means of screening hybridoma panels for antigen specificity. Nanodiscs were chosen to be tested in FACS over other membrane mimetic systems because they are considered to be an ideal method for the display of membrane proteins in a phospholipid setting with enhanced stability close to the native structure. Indeed, it has been shown using nanodiscs that unique phospholipid interactions affect native toxin binding interactions for the ion channel TRPV1.

They added "As a multi-pass membrane protein model for insertion into nanodiscs, we selected VSD4-NavAb to represent ion channels, a difcult antibody target and used hybridoma. The multi-pass membrane protein and tool hybridoma of the VSD4-NavAb model were used in this work to demonstrate the use of nanodiscs for cell sorting. The ability to integrate membrane proteins into nanodiscs may be target-dependent and must be determined empirically by screening the conditions of reconstitution. If available, we suggest the use of negative stain EM to track whether a homogeneous size distribution has been generated by the optimization process".

Nanodiscs provide a way for FACS to present membrane proteins in a soluble format that more closely mimics the structure of the native protein. The first author explain "We biotinylated the nanodisc for these experiments following the integration of the NavAb. While target protein labelling is widely used for FACS antigen-specific B-cells20-22, adding biotin to the antigen may lead to epitope modification recognised by specific antibodies. As we had previously found that 141B8 mAb was bound to NavAb, we were able to decide if the binding to its unique epitope was affected by biotinylation. If control antibodies or ligands are available for a target of interest, it is recommended that nanodisc incorporation and biotinylation using ELISA should be followed by binding". 

If control molecules are inaccessible and epitope diversity is required, before nanodisc formation, the MSP itself can be biotinylated, thus enhancing the concern of adding biotin to the epitope of interest on the target protein.

They showed that the integration of a multi-pass membrane protein into nanodisc membrane mimetics facilitates FACS sorting of antigen-specific hybridoma. The ability to positively classify antigen-specific requirements.

Hybridoma accelerates the detection of antibodies early in the antibody discovery process. Although it is not trivial to prepare validated nanodiscs that contain a target multi-pass membrane protein and require the precursor. 

The advantage of having a membrane protein in a stable form that is appropriate for tests used for soluble proteins justifies the impact of nanodisc production by having higher amounts of starting material and optimization time is needed for proteoliposome incorporation.