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. Author manuscript; available in PMC: 2023 Mar 6.
Published in final edited form as: Sci Immunol. 2022 Apr 29;7(70):eabj1640. doi: 10.1126/sciimmunol.abj1640

IgG3 hinge dependent enhancement of intracellular antiviral immunity

Stian Foss a,b,c,#, Alexandra Jonsson a,b,c,#, Maria Bottermann d, Ruth Watkinson d, Heidrun E Lode a,b,c, Martin B McAdam a,b, Terje E Michaelsen e,f, Inger Sandlie a,b,c, Leo C James d, Jan Terje Andersen b,c,✉
PMCID: PMC7614286  EMSID: EMS170879  PMID: 35486676

Abstract

Humans have four IgG antibody subclasses that selectively engage immune effector molecules to protect against infections. While IgG1 has been studied in detail and is the subclass of most approved antibody therapeutics, increasing evidence indicate that IgG3 is associated with enhanced protection against pathogens. Here we report that IgG3 has superior capacity to mediate intracellular anti-viral immunity compared with the other subclasses, which depends on its uniquely extended hinge region allowing favorable activation of the cytosolic Fc receptor and ubiquitin ligase TRIM21. TRIM21 may also synergize with complement C1/C4 mediated lysosomal degradation via capsid inactivation. We demonstrate that this process is also potentiated by IgG3 in a hinge dependent manner. Our findings reveal how the four IgG subclasses mediate intracellular immunity, knowledge that may guide IgG subclass selection and engineering of anti-viral antibodies for prophylaxis and therapy.

Introduction

Antibodies play a crucial role in the defense against bacterial and viral infections. They do so by either blocking host cell entry, inactivating pathogens by opsonization or by recruiting cellular effector functions via binding to Fc receptors (Klasse and Sattentau 2002, Bournazos and Ravetch 2017). In blood, IgG is the most prevalent isotype, which is further divided into four distinct subclasses, IgG1, IgG2, IgG3, and IgG4, in order of decreasing abundance (Morell, Skvaril et al. 1972). The co-evolution of IgG subclasses with pathogens has resulted in their interaction with soluble and cell bound effector molecules, as well as the neonatal Fc receptor (FcRn), which salvages IgG from intracellular degradation.

Despite sharing about 95% similarity in amino acid composition, the IgG subclasses have distinct differences that instruct selective engagement of effector molecules. In particular, there is variation in the sequence corresponding to the hinge region, which forms a linker between the two antigen binding Fab arms and the Fc region. Due to differences in length and amino acid composition, the flexibility of the hinge decreases in the order IgG3>IgG1>IgG4>IgG2 (Roux, Strelets et al. 1997, Carrasco, Garcia de la Torre et al. 2001). The hinges of IgG1 and IgG4 contain two disulfide bridges, there is four in IgG2, while IgG3 has a long hinge that encompasses up to 62 amino acids forming a polyproline helix with 11 disulfide bridges (Saluk and Clem 1971, Michaelsen and Natvig 1972, Johnson, Michaelsen et al. 1975, Michaelsen, Frangione et al. 1977). The elongated hinge of IgG3 enables high rotational freedom, which provides flexibility and reach. These properties may provide IgG3 with a functional advantage in activation of effector functions.

Although IgG3 constitutes only a minor proportion of total IgG in blood, recent studies have demonstrated that it plays a crucial role in protection against pathogens, including viruses, bacteria and parasites, as reviewed (Damelang, Rogerson et al. 2019, Chu, Patz et al. 2021). Interestingly, IgG3 responses have been shown to correlate with partial protection in a HIV vaccine trial (Katsinelos, Tuck et al. 2019). Furthermore, individuals with IgG3 subclass deficiency, but otherwise normal total IgG levels, tend to suffer from recurrent upper respiratory tract infections (Barton, Bertoli et al. 2016, Kim, Park et al. 2016). These data encourage structure-function studies of human IgG3 in the context of infectious diseases, knowledge that may be utilized in the design of therapeutics. So far, few monoclonal IgG antibodies have been approved for the treatment of infectious disease, but there is an increasing interest in exploring the potential of IgG3 for therapy and prophylaxis (Irani, Guy et al. 2015, Damelang, Rogerson et al. 2019, Chu, Patz et al. 2021). Moreover, the utility of monoclonal antibodies has further been highlighted by the ongoing pandemic of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (DeFrancesco 2020).

While extracellular IgG-mediated protection has been extensively studied, antibodies can also mediate protection in the cytosolic compartment of non-hematopoietic cells (Mallery, McEwan et al. 2010) as well as immune cells (Labzin, Bottermann et al. 2019, Ng, Kaliaperumal et al. 2019). This is due to engagement of the cytosolic antibody receptor and ubiquitin ligase tripartite motif containing-21 (TRIM21) that binds symmetrically to the CH2-CH3 interface of the Fc via its N-terminal PRYSPRY domain (James, Keeble et al. 2007) (Figure S1a-b). TRIM21 targets antibody-bound complexes for proteasomal degradation while at the same time activating a pro-inflammatory transcriptional program resulting in the production of cytokines and chemokines (Mallery, McEwan et al. 2010, McEwan, Tam et al. 2013, Bottermann, Foss et al. 2018) (Figure S1c). TRIM21 therefore functions as a last line of defense against pathogens that have breached extracellular defense mechanisms.

To fully understand the role of TRIM21 in intracellular protection, it is important to understand how the four human IgG subclasses trigger it’s anti-viral activity. Here, we report that IgG3 is the most potent in activating both effector function arms of TRIM21, followed by IgG1 and then IgG2 and IgG4. Importantly, we find that the IgG hinge region is deterministic of TRIM21-mediated anti-viral activity. Finally, we demonstrate that IgG3 also potentiates protection by restricting infection via complement C1/C4-mediated neutralization (Figure S1d) (Bottermann, Foss et al. 2019).

Results

TRIM21 binds human IgG subclasses equally well in vitro

To compare the four human IgG subclasses, we produced recombinant monoclonal mouse-human chimeric subclass variants (h9C12) with the variable domains derived from a hybridoma (9C12) (Figure 1a, Figure S2) (Foss, Watkinson et al. 2016). The antibody binds the hypervariable loops located at the apex of the hexon capsid protein of human adenovirus type 5 (Ad5), an immunodominant epitope (Varghese, Mikyas et al. 2004, Sumida, Truitt et al. 2005, Bradley, Lynch et al. 2012, Bradley, Maxfield et al. 2012, Bottermann, Lode et al. 2016). Purified h9C12 IgG fractions migrated according to their expected molecular weights in SDS-PAGE, IgG3 more slowly due to the long hinge, while a lower molecular weight band was observed for IgG4 (Figure 1b), in line with formation of half-molecules (Schuurman, Van Ree et al. 1999). All four h9C12 subclasses were further shown to bind equally well to the Ad5 hexon protein (Figure 1c) and to full-length TRIM21 when captured on hexon in ELISA (Figure 1d). TRIM21 binding was completely abolished for both IgG1 and IgG3 when the key interaction residue H433 was replaced with alanine (H433A) (Foss, Watkinson et al. 2016). In accordance with this, when a titration series of the C-terminal PRYSPRY domain of TRIM21 was injected over immobilized h9C12 subclasses using surface plasmon resonance (SPR), no major differences in KD values were measured (Table S1, Figure S3a-d).

Figure 1. IgG3 is a potent mediator of TRIM21 antiviral immunity.

Figure 1

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(a) Illustration showing the four human h9C12 IgG subclasses. The hinge region, Fab and Fc as well as the binding sites for TRIM21, FcRn, FcγRs and C1q in IgG1 are indicated. (b) Non-reducing SDS-PAGE showing migration of produced recombinant h9C12 IgG subclasses. (c) Binding of the h9C12 IgG subclasses (1000.0-0.45 ng/mL) to Ad5 hexon protein in ELISA. (d) Binding of Ad5 hexon captured h9C12 IgG subclasses, IgG1-H433A and IgG3-H433A (1000.0-0.45 ng/mL) to full-length TRIM21 in ELISA. Shown as mean±s.d of duplicates from a representative experiment. (e) TRIM21-mediated neutralization of Ad5-mCherry in complex with titrated amounts of h9C12 IgG subclasses (15.0-0.12 μg/mL). Shown as fold change between relative infection levels in WT and T21 KO 293T cells normalized to virus only, mean±s.d of triplicates from two independent experiments. ***p>0.001, ns=not significant by two-way ANOVA. (f) TRIM21-dependent NF-κB-induction in response to infection of WT 293T or T21 KO 293T reporter cells with Ad5 opsonized h9C12 IgG subclasses. (g) NF-κB-induction in response to infection of WT 293T reporter cells with h9C12 variants. Shown as fold change from virus only, mean±s.d of triplicates from a representative experiment, ***p>0.001, ns=not significant by one-way ANOVA.

IgG3 is a potent activator of TRIM21

Next, we compared the ability of the four h9C12 IgG subclasses to engage TRIM21 upon Ad5 infection of non-hematopoietic cells. To this end, we performed an in vitro neutralization assay in which WT or TRIM21 knockout (KO) 293T cells were infected with Ad5-mCherry in complex with titrated amounts of the antibodies. The 293T cells did not express surface FcγRs (Figure S4), in line with a previous report (Bottermann, Foss et al. 2019). Infection levels relative to that of Ad5-mCherry alone were determined by flow cytometry, and TRIM21-dependent neutralization calculated as fold change between infection levels in WT and TRIM21 KO cells. Surprisingly, we observed that IgG3 neutralized Ad5-mCherry more efficiently than IgG1 followed by IgG2 and IgG4 at both high and low levels of antibody (Figure 1e). No TRIM21-dependent neutralization activity was detected for H433A-containing IgG1 or IgG3. To exclude the possibility that aggregation or low pH stress of IgG3 was causing the enhanced neutralization phenotype, we repeated the experiment in WT cells comparing the stabilized IgG3 variant N392K/M397V (Saito, Namisaki et al. 2019) with IgG3, yielding near identical results (Figure S5). Finally, equimolar amounts of IgG1 and IgG3 bound equally well to Ad5 virus particles in ELISA (Figure S6).

TRIM21 antibody-dependent neutralization of Ad5 is followed by a wave of innate immune signaling (McEwan, Tam et al. 2013, Fletcher, Mallery et al. 2015, Bottermann, Foss et al. 2018). To address how the four IgG subclasses effect TRIM21-induced signaling, NF-κB reporter WT and TRIM21 KO 293T cells were infected with Ad5-mCherry complexed with h9C12 subclasses. Again, IgG3 showed superior activity, as it induced an over 2-fold more potent NF-κB response in WT cells compared to IgG1, IgG2 and IgG4, while no response was observed in TRIM21 KO cells (Figure 1f). Furthermore, the H433A substitution in IgG1 as well as in IgG3, fully abrogated NF-κB induction in WT cells upon infection, demonstrating that the response was entirely-dependent on TRIM21 and its interaction with antibody (Figure 1g). Thus, IgG3 is the most potent inducer of both arms of TRIM21 effector function during cellular infection, despite the fact that human IgG subclasses bind equally well to TRIM21 in biochemical assays.

The extended IgG3 hinge dictates enhanced TRIM21 activity

As the hinge of IgG3 is approximately four times as long as that of the other subclasses, we asked if this might be crucial for more potent TRIM21 activity. To address this, the hinge of IgG3 was replaced by that of IgG1, IgG2 and IgG4 and vice versa (Figure 2a-c; Figure S7). When the IgG3 hinge was introduced into IgG1 the S131C mutation was also included in the CH1 domain to ensure proper binding of the light chain. Similarly, the C131S mutation was introduced when the IgG1 hinge was introduced into IgG3. While the IgG1/IgG3 hinge swap variants bound equally well as their WT counterparts to the N-terminal PRYSPRY domain of TRIM21 (Figure S2a-c, Table S1), distinct differences in TRIM21-mediated Ad5 neutralization and especially NF-κB induction were measured in cellular infection assays. Specifically, IgG1 and IgG2 with the hinge of IgG3 showed increased TRIM21-dependent neutralization activity and NF-κB induction to the level of WT IgG3 (Figure 2a-b). A similar trend was observed for IgG4 with an IgG3 hinge, although the effect on neutralization was less prominent (Figure 2c). However, NF-κB induction was markedly more potent for IgG4-IgG3Hinge, as it approached the level of IgG3. Furthermore, replacing the hinge regions of IgG3 with that of the other subclasses reversed the neutralization and signaling phenotypes in all cases (Figure 2a-c). These results clearly demonstrate that structural differences between the IgG subclasses modulate TRIM21-dependent effector functions, and that the long hinge of IgG3 potentiates its anti-viral activity beyond that of the other subclasses.

Figure 2. IgG subclasses with a grafted IgG3 hinge potently activates TRIM21.

Figure 2

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Illustrations showing h9C12 hinge swap variants (left panel), TRIM21-mediated neutralization (middle panel) and NF-κB-induction (right panel) in response to infection of 293T cells with Ad5-mCherry in complex with (a) IgG1/IgG3, (b) IgG2/IgG3 and (c) IgG4/IgG3 h9C12 hinge swap variants. Neutralization data is presented as fold change between relative infection levels in WT and T21 KO 293T cells normalized to virus only, mean±s.d of triplicates from two independent experiments, ***p>0.001, **p>0.01, *p>0.05 and ns=not significant by two-way ANOVA. NF-κB data is presented as fold change relative to Ad5-mCherry only, mean±s.d of triplicates from representative experiments, ***p>0.001 and ns=not significant by one-way ANOVA.

Length and flexibility of the IgG3 hinge is pivotal for potent TRIM21 activity

The IgG3 hinge consists of 62 amino acids divided into 17-15-15-15mer segments (H1-H4) and held together by 11 disulfide bonds (Michaelsen, Frangione et al. 1977) (Figure 4a). In contrast, the IgG1 hinge consists of 15 amino acids and two disulfide bonds. The hinges of IgG2 and IgG4 have 12 amino acids and two or four disulfide bonds, respectively (Vidarsson, Dekkers et al. 2014). The upper hinge of IgG3 contains a stretch of 12 amino acids without any disulfide bonds that increase flexibility and reach of the Fab’s relative to the Fc (Roux, Strelets et al. 1997, Carrasco, Garcia de la Torre et al. 2001). In line with this, IgG3 was recently shown to have a greater spatial tolerance for antigen binding (Shaw, Hoffecker et al. 2019).

Figure 4. The IgG3 hinge enhances C1/C4-dependent Ad5 neutralization.

Figure 4

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(a) C1/C4-dependent neutralization of Ad5-mCherry in complex with h9C12 IgG subclasses or (b) IgG1, IgG3, IgG1-IgG3Hinge and IgG3-IgG1Hinge in HeLa cells in presence of C1q reconstituted human serum (1.0-0.008 μg/mL). (c) Time chase of C4 conversion in presence of Ad5-mCherry in complex with IgG1 and IgG3 or (d) IgG3-IgG1Hinge and IgG1-IgG3Hinge incubated in normal human serum. (e) C1/C4-dependent neutralization of Ad5-mCherry in complex with IgG4, IgG3, IgG4-IgG3Hinge and IgG3-IgG4Hinge, or (f) IgG3, IgG3ΔHI and IgG3ΔH2 in HeLa cells in presence of C1q reconstituted human serum (1.00.008 μg/mL). Neutralization data shown as fold neutralization compared to virus only and presented as mean ±s.d of triplicates from three independent experiments, ***p<0.001,*p<0.05 and ns=not significant by two-way ANOVA.

To address how these two structural features contribute to enhanced TRIM21 activity, we generated a panel of engineered IgG3 variants where the hinge was sequentially shortened by deletion of the exons encoding the four hinge segments, beginning at either H1 or H2 (Figure S8). Again, the antibodies were well produced and TRIM21 PRYSPRY binding was unaltered (Figure S2, Table S1). When evaluated for their ability to neutralize Ad5 via TRIM21 in 293T cells, we found that sequential removal of the hinge segments resulted in an overall reduction of neutralization activity, especially at low antibody concentration (Figure 3b-c). The effect was more prominent for NF-κB signaling than virus neutralization. This finding is in line with previous studies showing that alteration in antibody virus or antibody-TRIM21 affinity has a greater effect on signaling than neutralization (Bottermann, Lode et al. 2016, Foss, Watkinson et al. 2016). Moreover, hinge engineered variants lacking the first 17mer segment (H1) trended towards weaker neutralization and NF-κB induction at low concentrations of antibody compared to variants lacking the second 15mer hinge segment H2. This hinted to a role of IgG3 Fab flexibility, and we addressed the question by opening up the hinge by replacing the three first cysteines of the hinge with serine (Figure 3f, Figure S9). Interestingly, the IgG3-C1-3S variant gained both TRIM21-dependent neutralization and NF-κB induction activity beyond that of WT in the cellular infection assays (Figure 3g-h). These data demonstrate that the length and flexibility of the IgG3 hinge is the determinant for potentiated TRIM21 activity.

Figure 3. TRIM21 activity hinge on IgG3 hinge length and flexibility.

Figure 3

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(a) Illustration showing IgG3 and the amino acid composition of the four hinge segments (H1-H4), with cysteine residues involved in inter heavy chain disulfide bonds indicated in red. (b) TRIM21-mediated neutralization of Ad5-mCherry in complex with IgG3, IgG3ΔH1, IgG3ΔH1-2, IgG3ΔH1-2-3 (15.0-0.12 μg/mL) and (c) IgG3, IgG3ΔH2, IgG3ΔH2-3, IgG3ΔH2-3-4 (15.0-0.12 μg/mL) hinge truncated variants in 293T cells. (d) NF-κB-induction in response to infection of 293T reporter cells with Ad5-mCherry in complex with IgG3, IgG3ΔH1, IgG3ΔH1-2, IgG3ΔH1-2-3 or (e) IgG3, IgG3ΔH2, IgG3ΔH2-3, IgG3ΔH2-3-4 hinge truncated variants. (f) Illustration showing IgG3-C1-3S in which the three upper disulfide bonds of the hinge have been removed by mutating the corresponding cysteine residues to serine. (g) TRIM21-mediated neutralization in 293T cells of Ad5-mCherry in complex with IgG3 and IgG3-C1-3S (15.0-0.12 μg/mL). (h) NF-κB-induction in response to infection of 293T reporter cells with Ad5-mCherry in complex with IgG3 and IgG3-C1-3S. Neutralization data is presented as fold change between relative infection levels in WT and T21 KO 293T cells normalized to virus only, mean±s.d of triplicates from three representative experiments, ***p>0.001, **p>0.01, *p>0.05 and ns=not significant by two-way ANOVA. NF-κB data is presented as fold change relative to Ad5-mCherry only, mean±s.d of triplicates from representative experiments, **p>0.01, *p>0.05 and ns=not significant by one-way ANOVA.

IgG3 enhances complement C1/C4-mediated Ad5 neutralization

We have previously shown that rapid TRIM21-dependent degradation can work alongside antibody mediated complement C1/C4-dependent intracellular Ad5 neutralization (Bottermann, Foss et al. 2019). Here, C1/C4 is recruited to antibody coated Ad5 leading to covalent attachment of C4b to the virus. This results in capsid inactivation, prevents endosomal escape and increases lysosomal degradation (Figure S1d). To address the ability of the human IgG subclasses to neutralize Ad5 via complement C1/C4, Ad5-mCherry was incubated with h9C12 IgG subclasses in C1q-depleted human serum, reconstituted with titrated amounts of pure C1q and added to HeLa cells. As for 293T, HeLa cells did not express surface FcγRs (Figure S4). The number of infected cells was quantified by flow cytometry and relative infection levels were derived for each antibody variant compared to that of Ad5-mCherry only. The results showed that IgG3 was the most potent subclass, followed by IgG1 and IgG2, while no activity was observed for IgG4 (Figure 4a), mirroring the C1q binding hierarchy of the subclasses (Figure S10).

To address what effect the IgG3 hinge has on the C1/C4 neutralization mechanism, we compared the IgG1 and IgG3 hinge swap variants, which revealed that IgG1-IgG3Hinge was at least as potent as IgG3, while IgG3-IgG1Hinge phenocopied IgG1 (Figure 4b). Using a C4 convertase assay, in which IgG1 and IgG3 were pre-incubated with Ad5 before addition of human serum, measurements of C4 cleavage over time using western blot showed both more rapid and complete conversion of C4 into C4b in presence of IgG3 compared to IgG1 (Figure 4c). Importantly, comparing the activity of the WT antibodies to their hinge swap variants revealed that rapid and complete C4 conversion was indeed hinge-dependent (Figure 4d). However, IgG3Hinge introduced in IgG4 could not rescue C1/C4-mediated Ad5 neutralization (Figure 4e), nor did IgG4 induce C4 conversion (Figure S11). This shows that the IgG3 hinge potentiates intracellular C1/C4-dependent neutralization when linked to an Fc region with the ability to activate complement. Finally, we sought to determine if the upper 17mer hinge region of IgG3 influenced C1/C4-dependent Ad5 neutralization. To this end, we repeated the neutralization experiment using IgG3 variants with either the upper 17mer (△H1) or second 15mer (△H2) hinge region removed. The results showed that deletion of the 17mer hinge region reduced C1/C4-dependent neutralization to a much greater extent than when the second 15mer region was lacking (Figure 4f).

Taken together, the data demonstrate that human IgG3 induces superior intracellular TRIM21 mediated anti-viral immunity, and also C1/C4-dependent Ad5 neutralization in a hinge-dependent manner.

Discussion

As human IgG3 is the most potent subclass in the extracellular environment (Vidarsson, Dekkers et al. 2014), it plays an important role during initial stages of infection. Several studies support this by showing that IgG3 is an anti-viral subclass that is made acutely, and acts before an IgG1 response becomes dominant (Ferrante, Beard et al. 1990, Dugast, Stamatatos et al. 2014, Posadas-Mondragon, Aguilar-Faisal et al. 2017, Sadanand, Das et al. 2018). While a large body of evidence has provided in-depth insights into how the four human IgG subclasses interact with C1q, the classical FcγRs and FcRn to induce a range of distinct effector functions (Nimmerjahn and Ravetch 2008, Thielens, Tedesco et al. 2017, Pyzik, Sand et al. 2019), their contribution to intracellular host protection has so far not been studied in detail. Here, we demonstrate that the potent anti-viral activity of IgG3 extends into the cytosolic compartment via TRIM21, in a manner that depends on its uniquely long hinge region.

The IgG hinge region has been shown to influence extracellular effector functions. In a recent study, hinge-dependent enhancement of phagocytic activity was demonstrated for anti-HIV IgG1 and IgG3 hinge extended antibodies (Chu, Crowley et al. 2020). This is in line with data showing increased phagocytosis of bacteria bound by IgG3 rather than IgG1 (Vidarsson, van Der Pol et al. 2001, Goh, Grant et al. 2011). In contrast, agonistic anti-CD40 IgG’s that require Fc-FcγR co-engagement has been shown to benefit from the short rigid hinge of IgG2 rather than the long and flexible hinge of IgG3 (Liu, Zhao et al. 2019). In studies of complement activation, IgG3 has shown increased bactericidal activity over IgG1 against Neisseria when targeting a sparse antigen. IgG3 molecules with truncated or short artificial hinges increased complement activation and bactericidal activity independently of the density of target antigen (Michaelsen, Aase et al. 1990, Giuntini, Granoff et al. 2016). In our study, we demonstrate that enhanced TRIM21 effector activity triggered by IgG3 is dependent on its long and flexible hinge region. This was also the case for enhanced C1/C4-mediated neutralization.

Our results show that the capacity of the IgG subclasses to bridge virus recognition and cytosolic TRIM21 binding is an important feature. It determines the strength of the dual intracellular effector response, both of which are strictly dependent on the E3 ubiquitin ligase activity of TRIM21 (Mallery, McEwan et al. 2010, McEwan, Tam et al. 2013, Fletcher, Mallery et al. 2015). Recently, it was demonstrated that initiation of TRIM21 auto-ubiquitination requires a specific topology involving dimerization and a third catalytically active RING domain in close proximity induced by antibody clustering (Kiss, Clift et al. 2021, Zeng, Santos et al. 2021). Based on this, we hypothesize that a long and flexible hinge such as that of IgG3, may allow intermolecular TRIM21 RING domain contacts to form more readily (Figure S11). This is supported by our data showing efficient TRIM21 dependent neutralization by IgG3 at low antibody concentrations. Our data also suggest that the length and flexibility of the IgG3 hinge facilitates binding of C1q on the viral surface, and subsequent intracellular neutralization via the C1/C4 pathway.

Knowledge of the TRIM21 and C1/C4 neutralization mechanisms may well be utilized in the design of therapeutic interventions (Foss, Bottermann et al. 2019). While both mechanisms contribute to protective immunity, adverse effects have been described for Ad5-based gene therapy in which pre-existing antibody immunity leads to vector neutralization, innate immune signaling and block of transgene expression (Bottermann, Foss et al. 2018, Bottermann, Foss et al. 2019). These adverse effects may very well be exacerbated by IgG3. On the other hand, the potential of TRIM21 targeting is currently being explored for antibody treatment of neurodegenerative diseases targeting aggregated tau protein (McEwan, Falcon et al. 2017). In addition, TRIM21 can be used for selective degradation of intracellular proteins via the “TRIM-Away” technology, which also depends on RING domain clustering (Clift, McEwan et al. 2017, Clift, So et al. 2018, Zeng, Santos et al. 2021). Whether or not IgG3 would be advantageous for these purposes, for example to enhance degradation of protein with distally dispersed epitopes, remains to be explored.

Finally, TRIM21 has recently been identified as an activator of inflammasome formation in macrophages and to facilitate cross-presentation and activation of cytotoxic T cells against viral nucleoprotein in an antibody-dependent manner (Labzin, Bottermann et al. 2019, Ng, Kaliaperumal et al. 2019, Caddy, Vaysburd et al. 2020). Investigation into how the IgG subclasses work in concert with TRIM21 and the complement system in these settings should be studied to unravel their contribution. Such insights will surely guide antibody engineering for a range of indications.

Materials and methods

Cell culture

Adherent human embryonic kidney (HEK) 293E cells was maintained in RPMI-1640 medium (ThermoFisher) supplemented with 10% fetal bovine serum (ThermoFisher), 100 U/ml penicillin and 100 μg/ml streptomycin. HEK 293T and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, GlutaMAX) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were grown at 37°C in a humid 5% CO2, 95% air incubator.

Generation of TRIM21 knockout cells

HEK293T TRIM21 knockout cells were generated using CRISPR/Cas9 as described (Bottermann, Foss et al. 2019). Briefly, cells were seeded in 6-well plates (2.5 × 105 cell/well) and allowed to adhere overnight. Cells were then transfected with 1 μg pX458 (Addgene, USA) encoding TRIM21 gRNA. 24 hours post transfection cells were detached using trypsin and GFP positive cells were sorted into 96-well plates (1 cell/well) using FACS. On day 14 post sorting, individual clones were probed for TRIM21 knockdown using Sanger Sequencing (GATC Biotech).

Production of recombinant TRIM21

Full-length TRIM21 and TRIM21 PRYSPRY was expressed in BL21 derived Overexpress™ C41 (DE3) E.coli (Sigma-Aldrich) (James, Keeble et al. 2007, Clift, McEwan et al. 2017). Both proteins were purified from the cytoplasmic fraction using the Qiagen Ni-NTA Fast Start Kit. Monomeric fractions were isolated by size exclusion chromatography (SEC) using either a Superdex 200 Increase 30/100 GL or a Superdex 75 Increase 30/100 GL column (GE Healthcare), before up-concentration using 10K Amicon Ultra spin columns (Millipore). Protein concentrations were determined using a DeNovix DS-11+ spectrophotometer.

Production of rh9C12

Construction of expression vectors encoding recombinant rh9C12 antibodies has been described (Foss, Watkinson et al. 2016). Briefly, cDNA encoding the heavy chain of rh9C12 IgG subclasses were subcloned into the pLNOH2-hIgG1-WT-oriP vector (Norderhaug, Olafsen et al. 1997) using the BsmI/BamHI restriction sites (GenScript Inc). The expression vector encoding the rh9C12 κ light chain was made by cDNA synthesis and subcloned into the pLNOκ expression vector (Norderhaug, Olafsen et al. 1997). Single point mutations were introduced into the heavy chain vectors using site directed mutagenesis (GenScript Inc), while vectors encoding rh9C12 hinge swap and hinge deleted variants were made by cDNA synthesis (GenScript Inc) as cloned as above. rh9C12 subclasses and variants were produced in agherent HEK293E cells by transiently co-transfecting the heavy and light chain vectors using Lipofectamine 2000 (ThermoFisher). rh9C12 antibodies were purified using CaptureSelect™ pre-packed IgG-CH1 affinity columns (ThermoFisher). Size exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 column (GE Healthcare) was used to isolate monomeric rh9C12 fractions before up-concentration using 50K Amicon Ultra spin columns (Millipore). Protein concentrations were determined using a DeNovix DS-11+ spectrophotometer.

ELISA

Recombinant Ad5 hexon protein (BioRad) (diluted to 1 μg/ml in PBS) or replication deficient AdV5-GFP (SignaGen Laboratories) (diluted 1:1000 in PBS from a 1.0 x 1010 PFU/ml viral stock) was coated in 96-well plates (Corning Costar) and incubated overnight at 4°C. Remaining surface area was blocked using PBS/4% skim milk (S) (Sigma Aldrich), before washing four times with PBS/0.05% Tween 20 (T). Titrated amounts of h9C12 variants diluted in PBS/T/S were added to the wells and incubated for 1 h at room temperature (RT). Following washing as above, 6xHis-tagged full-length human TRIM21, each diluted to 1 μg/ml in PBS/S/T, was added and incubated for 1 h at RT. For C1q binding, human C1q (Complement Technologies) diluted to 0.366 μg/mL in veronal buffer containing 0.05% T was added and incubated for 30 min at 37°C. Following washing, captured rh9C12 was detected by an alkaline phosphatase (ALP)-conjugated anti-human κ-LC antibody produced in goat (Sigma-Aldrich) and bound 6xHis-tagged full-length human TRIM21 was detected by a mouse ALP-conjugated anti-6xHis antibody (Abcam). Bound human C1q was detected using a rabbit anti-hC1q antibody (Agilent Technologies) and an HRP-conjugated anti-rabbit antibody (GE Healthcare). Binding was visualized by addition of tetramethylbenzidine solution (Calbiochem) or ALP substrate (Sigma-Aldrich). The HRP-tetramethylbenzidine reaction was terminated by addition of 50 μl 1 M HCl, and the 450-nm (HRP) or 405-nm (ALP) absorption values were recorded using a Sunrise plate reader (TECAN).

TRIM21-mediated neutralization

HEK293T WT or T21 KO cells were plated at 5.0x104 cells/well in a 24-well plate and allowed to adhere overnight. The cells were cultured in serum-free media to ensure a complement free environment. Ad5-mCherry (ViraQuest) was diluted to 6 × 106 pts and mixed 1:1 with antibody (at the indicated concentration), and incubated for 30 min at RT to allow complex formation. 12 μL of the virus-antibody complex was added to WT or TRIM21 KO 293T cells in 500 μL DMEM per well and incubated for 24 hours at 37 °C. After infection, cells were collected by trypsinization, acquired on a BD LSRII flow cytometer (BD Biosciences, USA) and analyzed for mCherry gene expression using FlowJo® software (FlowJo LLC). TRIM21-dependent neutralization was calculated as fold change between RI in WT and TRIM21 KO cells.

C1q-mediated neutralization

Ad5-mCherry (ViraQuest) was diluted to 3 x 107 pts and mixed 1:1 with antibody (15 μg/mL) and allowed to complex for 30 min at RT. C1q depleted human serum was reconstituted with 100, 20, 4, 0.8 μg/mL human C1q (Complement Technologies) to a final dilution of 1:100, added to the Ad5-Ab mixture and allowed to incubate for 30 min at 37 °C. 16 μL was added to each cell culture well in 500 μL serum-free medium. Relative infection (RI) was calculated by dividing infectivity in presence of each rh9C12 variant by that of virus only treated cells.

NF-κB reporter assay

WT or TRIM21 KO HEK293T cells were transiently transfected with the pGL4.32 NF-κB luciferase reporter vector using Fugene 6 (both from Promega). 24 hours post transfection cells were plated at a density of 1 × 104 cells/well in clear-bottom 96-well plates (Corning Costar) and were allowed to adhere overnight. 3 × 1010 infectious units AdV5-mCherry/mL (ViraQuest) was mixed 1:1 with antibody (20 μg/mL), and incubated for 30 min at RT to allow for complex formation. 5 μL of the virus-antibody complex was added to each well and incubated for 7 hours at 37 °C before the addition of 100 μL steadylite plus luciferase reagent (Perkin Elmer) before analysis on a BMG PHERAstar FS plate reader.

Serum C4 Cleavage

Ad5 (ViraQuest) was diluted to 1x1011 pts/mL, mixed 1:1 with h9C12 WT and recombinant h9C12 variants (20 μg/mL) and allowed to complex for 30 min at RT. Human serum (Complement Technologies) was added to a final dilution of 1:60 and complexes were incubated for the indicated amount of time at 37 °C. Reactions were stopped by addition of 4x LDS and 10% dTT. C4 cleavage was analyzed by western blot.

Western blot

Samples were resuspended and heated at 95°C for 5 min in 4xLDS sample buffer with reducing agent (10% dTT). Equal volumes were loaded onto a 4-12% NuPAGE SDS-PAGE (ThermoFisher) gel and electrophoresed in 1xMOPS buffer (ThermoFisher). Proteins were transferred onto a nitrocellulose membrane (ThermoFisher) and immunoblotted with a mouse monoclonal anti-C4b (Abcam). In all cases, blots were incubated with Ab in PBS containing 5% milk and 0.1% Tween and washed with PBS/Tween. Visualization was carried out using an ECL Plus Western Blotting Detection System (GE Healthcare).

Statistical analysis

Data and statistical analysis was performed using GraphPad Prism (Version 9; GraphPad Software Inc). One or two-way ANOVA tests were performed with a 95% confidence interval where p<0.05 was considered a statistically significant difference.

Supplementary Material

Supplementary Figure

Footnotes

Conflict of Interest statement:

The authors declare no conflict of interest.

Acknowledgements

S.F., L.C.J. and J.T.A. designed research; S.F., A.J., and M.B., performed experiments; H.E.L., M.B.M., T.E.M. and I.S. contributed reagents and analytical tools; S.F., A.J., M.B., L.C.J. and J.T.A analyzed data and S.F., and A.J. made figures. S.F., and J.T.A wrote the paper. This work was supported by the Research Council of Norway through its Centre of Excellence funding scheme, Project 179573 (J.T.A., I.S. and S.F.), the grants 274993, 287927 (J.T.A.) and 251037/F20 (S.F.), and the South-Eastern Norway project 2018052 (J.T.A.).

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