Assessment of Fcγ receptor-dependent binding of influenza hemagglutinin vaccine-induced antibodies in a non-human primate model

Summary Several cross-protective antibodies that recognize a broad range of influenza A virus (IAV) strains are known to have functions in virus elimination such as Fcγ receptor (FcγR)-effector function and neutralizing activity against the head region. Although few studies have used primary cells as effector cells, the FcγR-effector function was evaluated after isolating each cell subset. Herein, we established an original assay system to evaluate purified FI6 IgG-mediated binding to hemagglutinin (HA)-expressing cells by flow cytometry using peripheral blood mononuclear cells from cynomolgus macaques. In addition, we evaluated the FcγR-effector function of IAV vaccine-induced anti-HA antibodies in cynomolgus macaques after administering the split vaccine. We found several cell types, mainly classical monocytes, bound to HA-expressing target cells in an FcγR-dependent manner, that were dominant in the binding of the cell population. Thus, this assay system could facilitate the development of a universal influenza vaccine.


INTRODUCTION
Current vaccines against seasonal influenza A viruses (IAVs) protect against infection by inducing neutralizing antibodies against the immunodominant region, which is predominantly the head domain of hemagglutinin (HA). IAVs often exhibit antigenic drift mainly in the head region, and highly pathogenic avian IAVs are considered a threat because they can cause pandemics because of their sporadic transmission to humans and resultant high mortality rates (Subbarao, 2018). Therefore, the protection efficacy of vaccines is reduced against not only pandemic strains but also antigen-mismatched seasonal IAV strains (Nelson and Holmes, 2007). Accordingly, there is an urgent need to develop a universal influenza vaccine that can induce effective immunity against a broad range of influenza virus strains, including not only seasonal IAV strains but also pandemic strains.
Various approaches have been used to develop universal influenza vaccines. One of the major attempts is the identification of cross-protective antibodies against broad IAV strains and their application for vaccine development by identifying the antigen epitope's regions recognized by these antibodies (Corti et al., 2017). In general, cross-protective antibodies demonstrate a broad spectrum of protection against infection by recognizing conserved epitopes that are poorly mutated. The candidate epitopes are the stem domain Corti et al., 2011;Tan et al., 2012), the receptor-binding site in the head domain (Shen et al., 2017;Whittle et al., 2011), and the lateral patch (Raymond et al., 2018) or vestigial esterase site (Bangaru et al., 2018). A broadly cross-reactive but non-neutralizing antibody targeting the trimer interface in the head domain has also been reported (Bangaru et al., 2019;Watanabe et al., 2019). These antibodies were mostly identified by the B-cell receptor sequences of B cells capable of recognizing a broad range of IAV strains in the blood after vaccination. In addition, one strategy could be to aim to provide this information to induce these cross-protective antibodies through vaccination, as has been reported in HIV (Jardine et al., 2015). Other attempts to design antigens have also been made. For example, it has been reported that immunization with HA bound to nanoparticles can induce cross-protective antibodies (Darricarrere et al., 2021;Kanekiyo et al., 2019).
To evaluate a developing vaccine, the hemagglutination inhibition (HAI) assay, the standard assay for estimating the efficacy of current IAV vaccines, is used for measuring the neutralizing activity against the influenza virus based on the binding capacity to epitopes around the receptor-binding site in the HA head region. However, it is not adequate to evaluate the overall function of antibodies that recognize a broad range of viral strains. Cross-protective antibodies are known to have Fcg receptor (FcgR) effector function in defense against IAV infection in vivo in addition to neutralizing activity, and the development of a novel assay system is required Bournazos et al., 2020;DiLillo et al., 2014DiLillo et al., , 2016Maamary et al., 2017).
There are three types of human Fc receptors, FcgRI (CD64), FcgRII (CD32), and FcgRIIIa (CD16), (Bruhns, 2012). In humans and monkeys, IgG1 and IgG3 readily bind to natural killer (NK) cells, neutrophils, monocytes, and macrophages, which express FcgRIIIa, whereas monocytes, macrophages, and dendritic cells express FcgRI and FcgRII Mullarkey et al., 2016;Seidel et al., 2013) and activate cells via FcgR, leading to antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (Boudreau and Alter, 2019). Several assays have been reported to evaluate FcgR-effector functions, such as ADCC and ADCP activities for specific cells such as NK cells, macrophages, and monocytes (Ana-Sosa-Batiz et al., 2016;Simhadri et al., 2015;Vanderven et al., 2016). Most of these utilize cultured cell lines as effector cells, and the mode of FcgR expression differs from that of primary cells. Furthermore, the FcgR-effector function is involved in the activation of CD8 + T cells in addition to NK cells, macrophages, monocytes, dendritic dells (Bournazos et al., 2020), and B cells (Maamary et al., 2017) rendering the evaluation of only specific cells as incomplete. Therefore, it is necessary to develop a system that can simultaneously evaluate the FcgR-effector function in primary cells. Moreover, for vaccine development, the selection of an animal model for vaccination is essential. The mouse is not a natural host for influenza, and influenza viruses that cause epidemics and pandemics in humans usually replicate inefficiently in the mouse unless an adapted virus is used. Furthermore, the subclasses of IgG and the expression pattern of FcR are known to differ from those in humans. Mice express a novel activating receptor, FcgRIV, which is functionally similar to human FcgRIIIa. However, mouse FcgRIV differs from human FcgRIIIa in distribution and IgG isotype binding (Bruhns, 2012). In mice, FcgRIV binds to IgG2a, IgG2b, and IgE, but not to IgG1 or IgG3. Non-human primate models such as rhesus and cynomolgus macaques were used to study the FcgR-effector function and showed that FcgR-mediated immune response-inducing antibodies are associated with reduced influenza virus shedding (Florek et al., 2014;Jegaskanda et al., 2013). Regarding FcgR, the distribution of FcgR expression is similar in humans and NHPs, making NHPs useful animal models for predicting FcgR function in humans. However, certain minor differences have been reported concerning the distribution of FcgR (Crowley and Ackerman, 2019). One of the differing aspects is the FcgRIIIb receptor, which contributes to the activation of granulocytes and is found on human granulocytes, while not present in macaque (Fossati et al., 2002).
In this study, we aimed to clarify the issues pertaining to the current vaccine by evaluating the antibodies induced by split vaccine administration in cynomolgus macaques and the associated FcgR-effector function in individual cells using peripheral blood mononuclear cells (PBMCs).

Generation of HA-expressing target cells
To quantitatively monitor the relative number of target cells, we first developed a cell line stably expressing the luciferase gene. Because the natural target of IAV was lung epithelial cells, the human lung epithelial A549 cell line was chosen as the parental cell line for the target cells. A549 cells were transduced with the luciferase gene under the control of the mouse retroviral long terminal repeat promoter (A549-luc). Because original A549 cells undergo cell death due to the spontaneous killing activity of NK cells when co-cultured with them, we selected an A549 clone that is resistant to their antibody-independent cytotoxic activity. For this, the A549-luc cells were first co-cultured with an NK cell line, KHYG-1, for one week and then co-cultured with another NK cell line, NK92 mi, for an additional week (total two weeks; Figure S1). We then cloned A549-NKR-luc cells as parental cells to establish cell lines stably expressing IAV hemagglutinin of H1 A/Puerto Rico/8/1934 (PR8-HA) and H1 A/California/07/2009 (CA-HA) cells.
Next, to establish a sensitive assay system to monitor the FcgR-effector function in non-human primate (NHP) cells, we modified FI6, which is known as a broad-spectrum anti-HA neutralizing monoclonal antibody (Corti et al., 2011), into a cynomolgus-type Fc region. We then generated FI6-cynoIgG1 and FI6-cynoIgG2 and purified them. The binding of cynoIgGs to HA on the cell surface of HA-expressing A549-NKR-luc cells was analyzed by flow cytometry. Both FI6-cynoIgG1 and IgG2 but not control iScience Article humanIgG1 bound to HA to the same extent and in a dose-dependent manner ( Figure 1 A and B). Therefore, these cynoIgGs were utilized to evaluate the Fc-effector functions in the following study.

Establishment of antibody-dependent binding assay
To evaluate whether these cynoIgGs can enable the cross-linking of HA-expressing target cells with immune effector cells in an FcgR-dependent manner, we used CFSE-labeled PBMCs and performed quantitative analysis by flow cytometry. First, we used CA-HA-expressing cells as target cells and target-effector binding was evaluated by the CFSE-positive population in the forward scatter high large-cell subset (Figure 2A). The binding of FI6-cynoIgG1 to target cells significantly increased the frequency of PBMC binding in a dose-dependent manner compared to control human IgG1 treatment ( Figures 2B and 2C).
Next, we examined whether the frequency of binding PBMCs bound was different for each IgG subclass. As a result, both FI6-cynoIgG1 and IgG2 significantly increased the frequency of binding cells compared to PBS treatment ( Figure 2D). However, in a comparison of IgG subclasses, FI6-cynoIgG1 showed significantly more PBMCs than IgG2 ( Figure 2D). The same trend was observed in PR8-HA-expressing cells ( Figure 2E). To confirm that the binding of PBMCs to target cells depends on antibody-FcgRs interaction, we generated FI6-cynoIgG1 harboring amino acid mutations L234A and L235A (LALA), which lacks the ability to bind to FcgRs in human and macaques (Lund et al., 1991;Parsons et al., 2019). When FI6-cynoIgG1 LALA was used instead of parental IgG1, the frequency of binding cells dramatically decreased to the same degree as in PBS ( Figures 2F and 2G), suggesting the requirement of Fc-FcgRs binding for the target-effector interaction observed in this assay. These results are consistent with previous reports from human IgG subclass Fceffector function assays (deTaeye et al., 2020; Vafa et al., 2014), suggesting that this assay could be utilized as a sensitive antibody-dependent binding assay using NHP samples.

Imaging flow cytometric analysis of binding cells
To confirm the FcgR-dependent target-effector binding, which was observed by conventional flow cytometry, we analyzed the binding using imaging flow cytometry, by which the multiplet status was visualized using fluorescent microscopy. To distinguish the target cells from effector PBMCs, A549-NKR-luc cells iScience Article were first marked with orange fluorescent-conjugated ER-Tracker, then the ER-Tracker labeled target cells and CFSE-labeled PBMCs were incubated in the presence or absence of FI6-cynoIgGs. Using imaging flow cytometry, ER-Tracker labeled target cells and CFSE-labeled PBMCs were detected as either singlets or multiplets ( Figure 3A). To visualize the multiplets on the scatter plot, plotting was performed with the intensity of CFSE (x-axis) and area (y-axis; an indicator of the cell size as FCS-A parameter in flow data) as in flow cytometry analysis (where target-effector doublets were defined by the FCS-A versus CFSE intensity). Consequently, the plot resembled that of flow cytometry (Figures 2A and 3B). For the quantification, the cells were divided into three regions (R1, R2, and R3). Region 1 (R1; large size and CFSE positive) indicated multiplets, Region 2 (R2; small size but CFSE positive) indicated CFSE-labeled PBMC singlet, and Region 3 (R3; large size but CFSE negative) indicated unbound target cells. To confirm whether R1 indeed contains the multiplets cells as the consequence of target-effector binding, we visually evaluated the mode of multiplets ( Figure S2) in each region carefully. We then counted the multiplets for each region ( Figures S2A and S2B) and found that the R1 had the most doublets in each region ( Figure 3C). When iScience Article the percentage of multiples was calculated for R1, the frequency of multiples was significantly higher in FI6-cynoIgG1 than in FI6-cynoIgG2 ( Figure 3D). This result was similar to that of the binding assay. These results indicate that the doublets detected by binding assay are the aggregates of effector cells bound to the target cells via the antibody-FcgR interaction.

FI6cynoIgG1 binds NK cells and classical monocytes to target cells via FcgRs
To investigate which immune cell subset in PBMCs is bound to the target cells, a staining panel for monkey PBMCs was designed to identify the expression of FcgRs and a subset of interest was created in the duplex gate ( Figure S3A). As shown in Figure 4A, treatment with FI6-cynoIgG1 resulted in the significant increase in the number of CD16 + CD32 + CD64 + , CD16 + CD32 À CD64 À , CD16 À CD32 + CD64 + , and CD16 À CD32 + CD64 À cells compared to that in PBS treatment. In contrast, treatment with FI6-cynoIgG2 increased the number of CD16 À CD32 + CD64 + cells as well as FI6-cynoIgG1, and the number of CD16 À CD32 À CD64 + cells was significantly increased compared to that in PBS treatment, which suggests that there is a wide variation in target-effector binding. Each CD16/CD32/CD64 single positive cell subset suggested the involvement of all three types of FcgRs for target-effector binding. Next, we investigated the cell phenotype of PBMCs bound to CA-HA-expressing target cells. We found that the target binding immune cells were mainly NK cells and classical monocytes ( Figures 4B and 4C). The same trend was observed for PR8-HA-expressing target cells (Figures S3B-S3D).
Taken together, these results suggest that the FcgR-dependent effector function induced by purified cyn-oIgG1 is likely mediated by NK cells and monocytes. We developed and validated an assay system to simultaneously evaluate various types of FcgR-mediated target-effector binding by using target cells with purified FI6 monoclonal antibodies and CFSE-labeled PBMCs. To evaluate the potential application of this assay for vaccine development, we examined whether our assay can be useful for analyzing FcgR-mediated target-effector binding by using sera from cynomolgus monkeys vaccinated in vivo. To obtain vaccine-induced anti-HA serum, monkeys were administered twice with the HA split vaccine against IVA CA according to the standard prime-boost regimen, and their blood was collected over time ( Figure S4A). Anti-HA antibody titers in the serum increased significantly after vaccination in all monkeys examined ( Figure 5A). Next, we performed a binding assay using sera from the sixth week after vaccination by diluting them according to the measured antibody titer and matching the amount of antibody bound to the target cells. Serum IgG were found to bind to the CA-HA-expressing target cells ( Figure S4B), although there was no correlation between the number of bound cells and antibody titer ( Figure 5B). Finally, the target-effector binding was examined ( Figure 2) and found that the number of CD16 À CD32 + CD64 + , CD16 + CD32 À CD64 + , CD16 À CD32 + CD64 À , and CD16 À CD32 À CD64 + cells significantly increased compared to that in PBS treatment, as observed in the case of FI6 ( Figure 5C).
Unlike FI6, the number of CD16 + CD32 À CD64 À cells was not increased by serum treatment. Coincidently, the subset of cells expressing FcgR was mostly classical monocytes ( Figures 5D and 5E). These results suggest that, at least in cynomolgus monkeys, the FcgR-effector function induced by the serum of the HA split vaccine is mainly mediated by classical monocytes.
To examine the involvement of FcRs other than FcgR, IgG in serum was purified and the binding assay was performed. The purified IgG in serum treatment showed the same trend as that of serum ( Figures 5F and  5G), which suggested that the binding PBMCs and target cells occurred mainly via anti-HA specific IgG. Furthermore, to examine the activation status of the binding classical monocytes, we assessed the induction of activation markers in the binding classical monocytes after binding to HA-CA target cells compared to that in unbound cells after serum treatment. The expression of CD80 mRNA was significantly upregulated, and CD83 mRNA was trended to be upregulated in the binding classical monocytes. It suggests that the classical monocytes were activated after binding to the target cells via anti-HA specific IgG ( Figure S5A).

DISCUSSION
In this study, we aimed to establish a PBMC-based assay system to evaluate FcgR-mediated target-effector binding, which potentially reflects the effector function of FcgR. The effector function of FcgR is critical for the development of a universal influenza vaccine. Most of the studies on ADCC and/or ADCP have used cell lines as the effector cells, and the presence or absence of cell-cell interaction has been ignored in cell-line based assays. However, in reality, there are multiple cells expressing FcgRs, and the effector cells exist as heterogeneous cell populations in the body. Thus, it is difficult to fully understand FcgR-mediated immune responses using cell lines. Therefore, we thought it was worth establishing an assay based on PBMCs, including multiple different immune cells, instead of a cell line.
However, it is advantageous to use the NHP model as an animal model to estimate the efficacy of vaccines, and the in vivo response after vaccine or antibody administration can be confirmed. Another important point in the use of NHP is that the expression distribution of FcgR is similar to that in humans. However, there has been no comprehensive assay system for the FcgR-effector function using the NHP model. Because we tested the system's applicability to non-clinical studies using the serum after administration of the H1N1 split influenza vaccine in cynomolgus macaques, the present study could be the first report to overcome these issues.
IgG1 is known to have a higher affinity than IgG2 for binding to each FcgR (Bruhns, 2012), and FI6-cynoIgG1 predominantly induces binding of CD16 + CD32 À CD64 À , CD16 À CD32 + CD64 + , and CD16 + CD32 + CD64 + cells to HA-expressing target cells. The binding of CD16 + CD32 À CD64 À cells to target cells is likely to occur via CD16. For CD16 À CD32 + CD64 + and CD16 + CD32 + CD64 + cells, binding to target cells is expected to occur mainly through CD64 because it has a higher binding capacity to IgG1 than CD32 and CD16, Therefore, all expressed FcgRs are functional for the binding. Furthermore, FI6-cynoIgG1-mediated binding utilizes NK cells and classical monocytes. However, NK cells may be included in CD16 + CD32 À CD64 À and classical monocytes in CD16 À CD32 + CD64 + cell populations. Thus, it is clear that a purified antibody iScience Article does not induce a single cell type; instead, multiple cell types bind to the target cells via the antibody and induce the FcgR-effector function. Although Expi293 was used to produce the recombinant FI6-cynoIgG used in this study, it is known that glycosylation differs depending on the type of cell line from which recombinant IgGs are produced. Glycosylation variation results in changes in the binding affinity and functionality of IgGs with FcRs (Boyoglu- Barnum et al., 2020;He et al., 2016;Jennewein and Alter, 2017). Therefore, it is unclear whether the glycosylation of the antibody in vivo can be directly reflected in this assay. Moreover, it has also been reported that ADCC induction is inhibited by high-titer HAI antibodies or the addition of an antibody with HAI activity to the serum of vaccines (Cox et al., 2016). Therefore, it is necessary to comprehensively evaluate antibodies in the serum induced by vaccine administration.
To comprehensively evaluate the antibodies that bind to HA in serum, we evaluated the FcgR-effector function of the current seasonal influenza virus vaccine using sera obtained after two doses of A/CA/07/2009 split vaccine in cynomolgus macaques. Because there was no correlation between the antibody titer and the number of PBMCs bound to the target cells, it is difficult to predict the FcgR-effector function based on the quantity of HAspecific IgG. NK cells were not well utilized as FcgR-mediated bound partners in sera of vaccinated cynomolgus macaques, unlike when purified FI6-cynoIgG1 was treated. Previous reports have shown that seasonal influenza vaccination does not sufficiently induce broadly reactive ADCC-induced antibodies, which is similar to the results of this study and suggests the usefulness of this evaluation system (Jacobsen et al., 2017;Jegaskanda et al., 2016;Vanderven et al., 2017). It has been reported that anti-influenza virus IgG monoclonal antibodies selectively bind to activated FcgRIIa to promote dendritic cell maturation and induction of CD8 + T cell responses (Bournazos et al., 2020), IgG produced by the inoculation may be involved in the FcgR-effector function via conventional dendritic cells, enhancing the efficacy of prevention and treatment of IAV infection.
It has been reported that trivalent influenza vaccination increases ADCP activity against the HA protein of the vaccine strain in adults (Ana-Sosa-Batiz et al., 2017). THP-1 cells, a monocyte cell line, were used in this study. We consistently demonstrated the involvement of monocytes in FcgR-mediated target-effector binding. Furthermore, detailed classification of monocytes revealed that among the three monocyte subsets, classical monocytes were mainly involved in binding. Because classical monocytes have been reported to have a higher phagocytic capacity than other monocyte subsets (Mukherjee et al., 2015), split vaccine administration is expected to bind classical monocytes to target cells via induced antibodies and exhibit ADCP activity. However, the level was the same as that of the treatment with FI6-cynoIgG1 purified antibody, which may be the limit of the ability of IgG to bind classical monocytes. Based on the above, we believe that ADCC activity can be enhanced by adding a Th1-type adjuvant that can induce NK cells to the current influenza split vaccine by adding purified antibody with high FcgR-effector activity or by changing the design of the antigen. This will lead to the development of a universal influenza vaccine. In addition, cross-reactivity with a broader range of HA strains could also be evaluated by changing the HA expressed by the target cells.
In conclusion, we developed an assay system to evaluate the FcgR-effector function using cynomolgus macaque PBMCs as effector cells and verified the effect using serum and PBMCs after split vaccine administration. The results showed that the antibodies induced by the split vaccine mediate FcR-dependent target-effector binding, and the main subset of the bound cell is classical monocytes. This assay system will be helpful for the development of a universal influenza vaccine because it will allow us to examine in detail the FcgR-effector function of individual cells induced by antibodies from passive immunization or vaccine administration in NHPs.

Limitations of the study
Because we analysis the binding activity in PBMCs in this study, we were unable to evaluate the FcgR effector function of blood cells such as neutrophils and other granulocytes and FcgR bearing cell in tissue like alveolar macrophages (He et al., 2017). We did not examine whether effector cells bound to the target cells are involved in the cytotoxicity, or phagocytosis of the target cells, and it will be essential to examine the function of effector cells in the future. In addition, as there is an inhibitory CD32B subtype of CD32, it is necessary to examine whether all bound cells have the FcgR-effector function.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  (Yamamoto et al., 2015). PBMCs were frozen in foetal bovine serum (FBS) containing 10% dimethyl sulfoxide and stored in liquid nitrogen until analysis.

Establishment of HA expressing NKR-luc cells
We first synthesized cDNA encoding firefly luciferase and cloned it into the pMx-IRES-Puro Vector (Cell Biolabs, Inc., San Diego, CA, USA). To prepare luc-transduced mouse retroviruses, pMx-fLuc-IRES-Puro was transfected into the packaging Platinum-A cells (Cell Biolabs, Inc.). After 48 h of transfection, the culture supernatants were collected and infected into A549 cells to obtain A549-luc cells. To establish target cells that are resistant to NK cell-mediated spontaneous cytotoxicity, we co-cultured A549-luc cells with NK92mi for one week and KHYG-1 for another week. As a result, we created an NK-resistant cell line, A549-NKR-luc.

Cynomolgus-sized monoclonal antibody
The pcDNA3.1-PD-1 cynoIgG1 heavy chain and the pcDNA6-PD-1 cynoIgG1 light chain vectors expressing the PD-1 cynoIgG1 heavy (GenBank: CS101552) and light chains (GenBank: AFF60184), respectively, were kindly gifted by Dr. Satoshi Nagata at the National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan. To generate a cynoIgG2 heavy chain constant region, a codon-optimized cynoIgG2 heavy chain constant region (GenBank: CS101554) fragment and cynoIgG1 LALA (L234A and L235A) were generated using a commercial gene synthesis service (GeneArt, Thermo Fisher Scientific, Waltham, MA, USA). The synthesized fragment was exchanged with cynoIgG1 constant regions in the pcDNA3.1-PD-1 cynoIgG heavy chain using NheI and NotI sites. Fv regions of the constructs were exchanged with FI6 heavy chain variable region (GenBank: JN234439) and light chain variable region (GenBank: JN234448) (Corti et al., 2011) fragments by the NEBuilder HiFi DNA Assembly (New England BioLabs, Ipswich, MA, USA).
FI6-cynoIgG1 and IgG2 were expressed using the Expi293 expression system (Thermo Fisher Scientific). Ex-pi293F cells were cultured in FreeStyle serum-free medium (Invitrogen, Carlsbad, CA, USA) and adjusted to 1 3 10 6 /mL in 100 mL FreeStyle serum-free medium. Each 100 mg of heavy chain and light chain plasmids and 100 mL 293fectin (Thermo Fisher Scientific) in 10 mL Opti-MEM (Thermo Fisher Scientific) were mixed and transfected into Expi293F cells. After 20 min, 100 mL FreeStyle serum-free medium was added, and the cells were incubated for 5dat 37 C in a 10% CO2 humidified atmosphere. The culture supernatants of the cells were collected and filtered through a 0.45-mm filter. The supernatant was concentrated using AKTA flux s (Cytiva, Tokyo, Japan) and diluted with an equal volume of 40 mM phosphate buffer (pH 7.0) (Nacalai Tesque, Kyoto, Japan). An automated protocol to purify cynoIgGs was established using HiTrap rProtein A FF columns (Cytiva) with AKTA Start (Cytiva). The solution was loaded onto a HiTrap rProtein A FF column equilibrated with 20 mM phosphate buffer. After washing with 20 mM phosphate buffer, FI6-cynoIgG was eluted with 100 mM glycine-HCl (pH 2.7) (Polysciences Inc., Warrington, PA, USA). Monitoring UV absorption at 280 nm facilitated fractionation and sample collection. After elution, the peak fractions were neutralized with 1 M Tris (pH 9.0) (Nippon Gene, Tokyo, Japan), dialyzed with phosphate-buffered saline (PBS) using Slide-A-Lyzer G2 Dialysis Cassettes (Thermo Fisher Scientific) at 4 C overnight. Finally, the antibody was concentrated at 1 mg/mL using an Amicon Ultra-3K (Thermo Fisher Scientific) (