2024 Volume 47 Issue 1 Pages 334-338
This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.
Antibodies play essential roles in the humoral immune system and are currently the core of biopharmaceuticals.1,2) The major design of therapeutic antibodies is based on the structure of the immunoglobulin G (IgG) molecule, which acts as a hub that mediates antigen binding through the Fab portions and interactions with the complement component C1 or Fcγ receptors (FcγRs) through the Fc portion, which leads to cytotoxic effector functions.3,4) Therefore, the functionality of therapeutic IgG antibodies is not limited to impeding virulent entities but includes the active elimination of pathogenic microorganisms and malignant cells. The mechanism of action of anticancer therapeutic antibodies is based on its effector functions, as exemplified by rituximab, a mouse/human-chimeric IgG1 that targets CD20 and is used for the treatment of specific non-Hodgkin lymphoma.5)
FcγRs consist of three major isoforms: high-affinity FcγRI, low-affinity FcγRII and FcγRIII, each of which is further divided into sub-isoforms, i.e., FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb.6–8) While FcγRIIIa on natural killer (NK) cells is responsible for antibody-dependent cellular cytotoxicity (ADCC), FcγRIIa on macrophages mediates antibody-dependent cellular phagocytosis (ADCP). Numerous attempts have been made to increase the affinity and specificity of therapeutic antibodies to these effector molecules and against specific antigens to improve their functionality.9,10) FcγRs and C1 share a common binding-site in the Fc region, which has been a major target for therapeutic antibody engineering through amino acid replacement and glycan remodeling.9) The IgG Fc portion possesses a conserved N-glycosylation site (Asn297) in each CH2 domain, and the N-glycan influences its interaction with FcγRs and C1 and the subsequent effector functions.11) In particular, fucosylation of the N-glycan decreases the affinity of IgG for FcγRIIIa, thereby compromising ADCC, which can thus be enhanced by afucosylation.12,13) Mogamulizumab is a typical afucosylated therapeutic IgG1 antibody, which targets the C-C chemokine receptor 4 (CCR4) and is used to treat adult T-cell leukemia/lymphoma.14)
In the development and evaluation of therapeutic antibodies, it is of paramount importance to quantitatively assess their interactions with the effector molecules. To achieve this, kinetic measurements based on surface plasmon resonance, optical interferometry, and microcalorimetric titration have been widely utilized.15–17) Generally, these bulk methods observe the collective behavior of numerous molecules in a solution, thereby providing averaged information about them. However, it should be noted that under certain solution and storage conditions, and due to mutations, IgG molecules might inevitably aggregate.18) Even a small amount of these aggregates can potentially influence the observed outcomes through these bulk-based techniques. This is primarily because the apparent affinity of immobilized effector molecules or inherently multivalent effector molecules, such as C1 becomes considerably augmented due to the presence of multiple binding sites within the aggregates.19)
Recently, advanced single-molecule observation techniques have emerged that can precisely analyze protein–protein interactions.20) Notably, high-speed atomic force microscopy (HS-AFM) facilitates the real-time, label-free observation of biomolecular interactions at the single-molecule level, and concurrently offers insights into the shape and size of the interacting molecules.21,22) In previous studies, we employed this technique to observe the stoichiometric interactions of IgG molecules with FcγRIIIa and C1.23–25) In this study, we explore the applicability of the HS-AFM technique to quantitatively evaluate the interactions of therapeutic IgG molecules with low-affinity FcγRs, i.e., FcγRIIa and FcγRIIIa, with rituximab, mogamulizumab, and their Fc fragments as test molecules.
We prepared the extracellular domains of FcγRIIa and FcγRIIIa with a C-terminal hexahistidine moiety using bacterial and mammalian expression systems, respectively. These soluble forms of FcγRs (sFcγRs) were placed onto a Ni2+-coated mica surface. In the observation buffer of the HS-AFM sample chamber, we added the solutions of the analytes, i.e., rituximab, mogamulizumab, and the Fc fragments derived from these therapeutic antibodies. We then performed real-time observation of their interactions with the FcγRs and measured the dwell times of the individual analytes, while confirming that the analytes underwent these interactions as single molecules without the formation of aggregates. We quantified the interactions of the analyte molecules based on the HS-AFM imaging (Figs. 1, 2, Supplementary Movies 1–8) by measuring the dwell times of the individual molecules. The measured dwell times within the observed time frame were cumulatively added to create a graph, from which a constant time (τ), corresponding to the reciprocal of the dissociation rate constant (koff) in a simple bimolecular reaction, was calculated26) (Fig. 3).
Clipped HS-AFM images visualizing the time-dependent binding of IgG (mogamulizumab in this figure) to the immobilized FcγR (FcγRIIa in this figure). IgG molecules that appeared in the frame are shown with white dotted circles, while those that remained from the former frame are shown with red dotted circles. Imaging rate, 0.2 s/frame. Scale bar, 30 nm.
Clipped HS-AFM images are shown for the binding of (A) rituximab and (B) mogamulizumab to FcγRIIIa. The images were captured after sufficient time had passed (> 1 min) after the addition of the antibody to the observation solution, to allow the binding-dissociation process to reach equilibrium. The IgG molecules bound to FcγRIIIa are shown with white solid circles. A greater amount of mogamulizumab bound to FcγRIIIa as compared to rituximab. Imaging rate, 0.2 s/frame. Scale bar, 30 nm.
Integrated number of the dwell times of the IgG molecules on (A) the FcγRIIIa- and (B) FcγRIIa-coated mica surfaces are shown for rituximab, rituximab-Fc, mogamulizumab, and mogamulizumab-Fc.
Our results revealed that mogamulizumab bound to FcγRIIIa with a 2–3-fold greater τ compared to that of rituximab in both the full-length and Fc forms (Figs. 2, 3). This is consistent with previous data obtained through biolayer interferometry, which indicated that rituximab showed a higher koff than mogamulizumab to FcγRIIIa.27) We confirmed that mogamulizumab mostly (>99.3%) lacked core fucosylation, while rituximab used in the present study was fully (>98.7%) fucosylated. These findings suggest that the absence of core fucose from the Fc-linked N-glycans contributes to the stabilization of the complex formed with FcγRIIIa, which results in an enhanced ADCC response.12,13) In either case, the Fc fragment showed a shorter τ than the full-length form. This is also consistent with previous data, which suggests that the Fab portions contribute to the interactions with FcγRIIIa of these antibodies.23,27)
Our data also indicated that rituximab and mogamulizumab exhibited comparable τ values with FcγRIIa, which is consistent with a previous report showing that the IgG1-Fc glycoform had little to no effect on koff and KD in the interaction with FcγRIIa: The core fucosylation had no significant influence on the interaction.28) It should be noted that, as in the case of FcγRIIIa, the full-length forms of IgG1 exhibited greater τ values with FcγRIIa compared to that of its Fc fragment, which indicates the contribution of the Fab portions to the interaction with FcγRIIa.
Our HS-AFM approach provided quantitative insights into the interactions of therapeutic antibodies with FcγRs, thus offering a detailed representation of the duration and frequency of an antibody molecule remains bound to an FcγR. This analysis aligns with the concept of ‘kinetic proofreading,’ where cells demonstrate the ability to discern ligands based on their variable residence times on the cell surface receptors.29,30) This selective discrimination arises from the requirement of a specific duration for the triggering of events within a ligand-activated receptor complex, which is disrupted if the ligand prematurely dissociates. Therefore, the observation of the dwell times on effector molecules at the single-molecule level serves as a valuable indicator to evaluate the efficacy of therapeutic antibodies. Although a laboratory-built HS-AFM instrument was used in the present study, commercially available instruments are becoming more widely used and the methods presented here can be carried out using these conventional devices. In parallel, the HS-AFM method synergistically complements the evaluation of the antibody-FcγR interactions alongside the promising emergent mass photometry technique, which provides the binding constants of the biomolecular complexes at the single-molecule level,31,32) thus further enhancing our understanding of these interactions.
The present HS-AFM data indicate that the Fab portion of IgG1 harbors a certain subsite for interaction with FcγRIIa and FcγRIIIa, which enhances the interaction through their canonical binding site in Fc. Moreover, Fab may play a role in conveying allosteric effects on the Fc conformation, and experimental and theoretical exploration of the IgG1 dynamic structure will be necessary to clarify these aspects. The findings in this line of study will contribute to the development of therapeutic antibodies that have been modified to enhance or diminish interactions with specific FcγRs.
We purchased rituximab from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), and mogamulizumab from Kyowa Kirin Co., Ltd. (Tokyo, Japan). Glycosylation profiling was performed by subjecting Fc (0.1 mg) to HPLC-based N-glycosylation profiling as previously described.33,34) The pyridylamino (PA) derivatives of oligosaccharides released from Fc were applied to a Shim-pack HRC-octadecyl silica (ODS) reverse-phase HPLC column and identified based on their elution times.
These IgG1 antibodies were purified by gel filtration using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, Chicago, IL, U.S.A.) with a 50 mM Tris–HCl, pH 8.0, buffer containing 150 mM NaCl. For the preparation of Fc fragments, the antibodies were dissolved at a final concentration of 10 mg/mL in 75 mM phosphate buffer, pH 6.0, containing 75 mM NaCl and 2 mM ethylenediaminetetraacetic acid, in the presence of papain (Merck, Darmstadt, Germany) with an enzyme/substrate ratio of 2% at 37 °C for 12 h. The reaction was terminated by adding 33 mM N-ehylmaleimide. The reaction mixture was applied to an nProtein A Sepharose Fast Flow (GE Healthcare) for the isolation of the Fc fragments. The Fc fragments were further purified by gel filtration using a Superdex 200 Increase 10/300 GL column (GE Healthcare).23) For the mock-treated controls, we incubated each IgG1 under the same conditions except for the absence of papain. Purity of each sample protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Preparation of Recombinant FcγRsThe cDNA that encodes the extracellular region of human FcγRIIa, residues 1–173 of the mature sequence, was cloned into a pET-21a vector (Novagen). The DNA sequence corresponding to the C-terminal hexahistidine tag (Ala-Ala-Ala-Leu-Glu-His6) was added to the 3′ end of the gene to facilitate purification. Using this construct, the human FcγRIIa with a C-terminal hexahistidine tag was expressed as an inclusion body in Escherichia coli BL21 cells grown in Luria–Bertani medium. The protein was refolded in vitro according to the previously reported protocol for the refolding of sFcγRIIIb.35) A mutationally deglycosylated sFcγRIIIa retaining only two N-glycosylation sites, i.e., Asn45 and Asn162, was produced with the C-terminal hexahistidine tag in Chinese hamster ovary cells as previously described.23,36) These sFcγRs were purified using cOmplete His-Tag Purification Resin (Roche, Basel, Switzerland) and then further purified on the HiLoad 26/60 Superdex 75 pg column (Cytiva, Marlborough, MA, U.S.A.).
HS-AFM Observation and AnalysisWe utilized a laboratory-built HS-AFM instrument, operating in tapping mode at room temperature. This system utilized a small cantilever with the following properties: a resonant frequency of approximately 0.6 MHz in a water environment; a spring constant of approximately 0.2 N/m; and a quality factor of roughly 2 at the resonant frequency. For additional details on the equipment, see the following paper.37)
To immobilize sFcγRs using their C-terminal hexahistidine tag, we treated the freshly cleaved mica surface (Muscovite mica, V1 grade, 1.5-mmφ) by applying a droplet of 10 mM NiCl2. After incubation for 3 min, the mica surface was thoroughly washed with Milli-Q water. Subsequently, an approximately 2 µL droplet of sFcγR solution (3 mg/mL) was placed on the surface and incubated for 5 min. The unbound sFcγR molecules were removed by rinsing with the observation buffer (50 mM Tris–HCl buffer at pH 8.0, containing 150 mM NaCl). Following this washing step, the HS-AFM sample stage was immersed in a chamber filled with approximately 70 µL of the observation buffer. Following the verification of sFcγR coverage on the mica substrate through HS-AFM imaging, the antibody was introduced into the observation solution. After the addition of the antibody, the observation solution was carefully pipetted to ensure the homogeneous distribution of the antibody molecules within the solution at a final concentration of 1.5 µg/mL for IgG or 180 µg/mL for Fc. The dwell time analysis was performed on the data after the antibody-sFcγR interaction had reached sufficient equilibrium, typically 1 min after the addition of the antibody and pipetting.
We measured the dwell times of the individual analyte molecules on sFcγR using successive HS-AFM images by monitoring their interactions as appearance or disappearance of bright spots on the images. At the IgG concentrations described above, the binding and dissociation of IgG to FcγR could be observed at the single molecule level. Because the Fc fragment has a shorter dwell time than the full-length IgG, the concentration was set higher to accurately estimate the dwell time. To examine potential non-specific FcγR interactions, we observed bovine serum albumin (Sigma-Aldrich, St. Louis, MO, U.S.A., 290 µg/mL) instead of IgG/Fc as a control. This control did show not any discernible binding with either FcγR (Supplementary Movie 10), confirming the specificity of the observed FcγR interactions with IgG/Fc. To quantify the interactions between the various combinations of IgG, Fc, and sFcγRs, we computed τ based on the binding dwell time. To determine τ, we assumed a single exponential function for the dwell time distribution. Rather than generating a histogram with fixed bin sizes, we constructed a graph illustrating the cumulative count of the dwell times by summing the data points reflecting an increase in the dwell time.26) The τ value was derived by fitting this graph of cumulative numbers against the dwell times using the integral of an exponential function. All analyses were performed using custom software developed with IgorPro 9 (WaveMetrics, Inc., Lake Oswego, OR, U.S.A.).
This work was supported in part by MEXT/JSPS Grants-in-Aid for Scientific Research (Grant Nos.: JP19J15602 to R.Y., JP17H05893, JP20K15981, and JP22H02755 to S.Y., and JP19H01017 to K.K.), and AMED (Grant No.: JP21ae0121020h0001 to S.Y.). We thank Yukiko Isono (Institute for Molecular Science) for her help in the preparation of the recombinant proteins, and Prof. Katsumi Maenaka (Hokkaido University) for useful discussions. Part of this study was conducted under the Human Glycome Atlas Project (HGA). This work was also supported by the Joint Research of the Exploratory Research Center on Life and Living Systems (ExCELLS) (ExCELLS Program Nos. 18-101 to T.U. and 23EXC601 to K.K. and T.U.) and by MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program (CURE) Grant Number: JPMXP1323015482.
The authors declare no conflict of interest.
This article contains supplementary materials.
Mesayamas Kongsema (Deceased on 5th January, 2021)
