INTERACTION CHARACTERIZATION BETWEEN THE SDAB-MRH-IGG NANOBODY AND IMMUNOGLOBULIN G

Nanobodies, also known as VHHs, are unique single variable domains from heavy-chain antibodies that contain the site for antigen binding. These antibody globular domains (12-16 kDa) can be efficiently made in Escherichia coli because they do not need complex pos-translational processing, which lowers production costs. Nanobodies were first found in camelids in the 1990s [1]. 

Nanobodies stand out because they are very stable in their conformation and are highly specific. They also have high aqueous solubility, which is linked to having more hydrophilic amino acids compared to human IgGs [2]. These features give nanobodies great potential for biotechnology, including use as biopharmaceuticals and as molecular sensors in diagnostic tests [3]. 

In 2018, Pleiner and co-workers created a library of VHHs by immunizing alpacas with mouse and rabbit IgGs, then selected them using phage display [4]. Among the ones found, sdAb-mrh-IgG was chosen because it recognizes the Fc region of mouse, rat, and human IgGs, increasing its possible uses in therapy and diagnosis. 

Even though the Protein Data Bank has 395 structures for 151 nanobodies, there is still no structural data for complexes between VHHs and IgGs. The goal of this study was to describe how sdAb-mrh-IgG interacts with a human IgG. Understanding the 3D structure of the sdAb-mrh-IgG–IgG complex is key to knowing how they recognize each other at a molecular level, and it will help in designing new interactions using computational tools. 

For this study, the nanobody sdAb-mrh-IgG was produced in E. coli BL-21 (DE3) strain. The sequence encoding the nanobody was cloned into a pET-25(b)+ expression vector, containing a His-tag purification sequence and an ampicillin selection marker. For NMR analyses, the protein was expressed in M9 medium with ¹³C-glucose and ¹⁵NH₄Cl, at 37 °C, for 18 hours. Purification involved separating the cell mass by centrifugation, followed by sonication lysis and filtration through a 0.45 μm membrane. Two chromatographic steps were then employed: nickel affinity and size exclusion. The sample was concentrated using a 3 kDa cut-off membrane centrifugal device, and the final protein concentration was 0.49 mM.  

The oligomeric state of the nanobody was assessed through analytical gel filtration, which confirmed its presence predominantly in a monomeric state. Its thermal stability was subsequently evaluated using a thermal denaturation process, monitored by circular dichroism (CD) spectroscopy. This analysis revealed a melting temperature (T_m​) of approximately 70.5 °C. Furthermore, circular dichroism was also employed to determine the nanobody's secondary structure composition, which indicated a major proportion of β-strand elements. 

The interaction of the nanobody with human IgG was characterized using complementary biophysical techniques. Surface Plasmon Resonance (SPR) analysis determined an affinity (K​_D) of 0.24 µM, which was noted as relatively low when compared to affinities reported for other nanobodies. This interaction was also observed by Nuclear Magnetic Resonance (NMR) spectroscopy. 2D [¹H, ¹⁵N]-HSQC spectra were acquired on an Avance III 700 MHz spectrometer (Bruker Biospin) across a range of temperatures: 298 K, 303 K, 313 K, 323 K, and 333 K. The primary objectives of these NMR acquisitions were twofold: first, to determine the temperature coefficient of the nanobody in the absence of any ligand; and second, to characterize its molecular mechanism of interaction in the presence of 14.8 µM human IgG (Sigma-Aldrich), including the determination of the temperature coefficient specific to this interaction. The precise determination of the temperature coefficient is crucial as it allows for direct inferences regarding the conformational stability of the protein and any structural alterations induced by its interaction with the ligand (IgG). In addition, the relative intensity of each backbone amino acid was individually evaluated to discern differences between the free and bound sdAb-mrh-IgG states. To further elucidate the conformational dynamics of this protein, additional NMR spectra were collected for the quantification of heteronuclear NOEs and for the measurement of relaxation parameters (T1​ and T2​). All obtained NMR spectra were processed using TopSpin 3.6.3 software. 

Our results indicate that the sdAb-mrh-IgG nanobody exhibits a highly stable 3D structure as indicated by conformational dynamics from NMR and circular dichroism data. Detailed NMR analyses of its interaction with human IgG indicate intricate mechanism of interaction which might involve allosteric conformational changes after binding in the studied temperature range.