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Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

T here are four different subclasses of IgG. The isotypes are highly homologous yet play distinct roles in the immune response (1)(2)(3). The lower hinge region, directly adjacent to the fragment crystallizable (Fc) region, has been found to be crucial for the interaction of IgG with both FcgRs and C1q. FcgRs mediate immune effector functions such as Ab-dependent cellular phagocytosis and Ab-dependent cellular cytotoxicity (ADCC).
Human FcgRs can be divided into activating and inhibitory receptors. Activating receptors are FcgRI, FcgRIIa, and FcgRIIIa, of which FcgRI has by far the highest affinity for IgG. In contrast, FcgRIIb mediates inhibitory effects. FcgRs are widely expressed on myeloid cells, including neutrophils, monocytes, and NK cells and facilitate Fc-dependent effector functions such as Ab-dependent cellular phagocytosis and/or ADCC against IgG-opsonized targets (6,9).
C1q is the first component of the classical complement pathway. It binds IgG with very low affinity and only becomes activated by recognizing multiple aggregated IgG molecules bound to a target surface, eventually leading to the formation of the membrane attack complex (MAC) C5-9. The MAC disturbs membrane integrity and eventually promotes cell lysis (14). C1q binding to IgG has been reported to mainly depend on amino acids in the lower hinge and CH2, largely overlapping regions required for FcgR binding (1,(15)(16)(17). In this study, IgG1 and IgG2 show clear distinction, as IgG1 binds and activates complement very efficiently, whereas IgG2 does less so and requires higher concentrations and/or epitope densities but can also effectively activate complement if a target has repetitive epitopes such as found on bacterial polysaccharides (1,17,18).
Human IgG is also recognized by the neonatal Fc receptor (FcRn), responsible for salvaging of IgG from lysosomes and therefore for the long half-life of IgG. The same receptor system is also responsible for transcytosis and transplacental transport of IgG from mother to fetus (19)(20)(21). We recently reported that this process is negatively affected for IgG2 in comparison with IgG1 by the lack of G236 in IgG2, which may explain the relatively low placental transport rates of IgG2 compared with IgG1. This low transport efficiency could be transferred to IgG1 by deletion of G236 (IgG1ΔG236). Vice versa, insertion of G236 into IgG2 (IgG2+G236) increased placental transport to levels of IgG1 (22,23). As glycine is devoid of side chains, making it extremely flexible in proteins, we hypothesized that the presence of G236 is important for increased Fab flexibility, which affects transport (22).
G236 is located in the lower hinge region of IgG, which is a region generally important for FcgR and C1q binding. The impact of mutations encompassing the deletion of G236 of IgG1 and the insertion of G236 to IgG2 have been partly studied regarding FcgR binding and functions (17,(24)(25)(26)(27)(28). The G236A substitution in IgG1 has been reported to increase binding to FcgRIIa and FcgRIIa-mediated phagocytosis (29). Variants of IgG1 expressing alanine at the adjacent position 237, normally a glycine in all IgG subclasses (G237A), has been reported to have reduced FcgRI affinity and reduced FcgRIIIa-mediated ADCC as well as CDC (17). However, these publications only partially distinguished between different FcgR polymorphisms and did not, except for Morgan et al. (17), investigate the impact on complement activation.
Based on our findings of this region on FcRn-mediated transport (22,23), we revisited these questions and functionally characterized the influence of substitutions and deletion at positions 236 and 237 in IgG1 and IgG2, this time for all FcgRs and their polymorphic variants as well as complement. Thereby, we want to dissect the role of the glycines in the lower hinge on the various Fc-dependent effector functions and identify new Fc-engineered variants with a specific effector function profile.

Ethics
Peripheral blood from anonymous, healthy volunteers was obtained with informed, written consent of all subjects in accordance with Dutch regulations. This study was approved by Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki.

Cells
Human embryonic kidney (HEK) 293F cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific) at 37˚C at 5% CO 2 and shaking at 125 rpm.
RAMOS cells were obtained from American Type Culture Collection (ATCC; ATCC CRL-1596) and grown in RPMI 1640 with 10% FCS at 37˚C at 5% CO 2 .
NK cell isolation was performed as described elsewhere (13). In short, freshly drawn heparinized blood was diluted 1:1 with 13 PBS containing 10% trisodium citrate (TSC) (v/v). Twenty-five milliliters of diluted blood were layered on 15 ml Ficoll-Paque PLUS (GE Healthcare). Fractions were separated at 400 3 g for 30 min at room temperature (RT) without break. Ring fractions of the same donor were combined and washed twice with 13 PBS containing 10% TSC. After counting cells, PBMCs were resuspended in 85 ml of 13 PBS containing 10% TSC and 0.5% human serum albumin (Cealb; Sanquin) (MACS buffer) and 15 ml of CD56 MACS beads (Miltenyi Biotec) per 1 3 10 7 cells. After 15 min on ice, cells were washed twice in MACS buffer, applied to an MS Column (Miltenyi Biotec), washed three times, and eluted. Cells were resuspended in IMDM (Life Technologies) containing 10% FCS and incubated at 37˚C at 5% CO 2 .
Polymorphonuclear neutrophils (PMNs) were isolated from freshly drawn heparinized blood. Blood was diluted 1:1 with 13 PBS, and 25 ml of diluted blood was layered on 15 ml of Ficoll-Paque PLUS (GE Healthcare). Fractions were separated at 400 3 g for 30 min at RT without break. After discarding the top layers, the RBC pellet containing the PMNs was lysed with hypotonic lysis buffer (30) on ice for 15 min. Cells were spun down at 290 3 g, the supernatant was carefully discarded, and the pellet was resuspended in 10 ml of lysis buffer. After 5-min incubation on ice, cells were washed with 13 PBS, resuspended in IMDM (Life Technologies) with 10% (v/v) FCS.
To increase FcgRI expression, PMNs were stimulated overnight with 50 ng/ml IFN-g (Sigma-Aldrich) and 10 ng/ml G-CSF (Sigma-Aldrich) at a cell concentration of 5 3 10 6 cells/ml in IMDM (Life Technologies) with 10% (v/v) FCS. To determine FcgRI expression, PMNs were stained with anti-CD64 biotin (Becton Dickinson) in 13 PBS with 0.1% (m/v) BSA for 30 min on ice, followed by 15 min with streptavidinallophycocyanin (BioLegend) after washing twice under the same conditions.
Purified RBCs were kindly provided by the Department of Erythrocyte Serology, Sanquin. For RBC biotinylation, 1.4 mg EZ-Link NHS-PEG12-Biotin (Thermo Fisher Scientific) was used to biotinylate 11 3 10 7 RBCs in a total volume of 250 ml for 15 min. Reaction was quenched by washing cells twice with IMDM (Life Technologies) containing 10% (v/v) FCS.
RBCs were labeled using a PKH26 Red Fluorescent Cell Linker Kit (Thermo Fisher Scientific) following manufacturer's instructions.

Cloning of Fc variants in anti-biotin and anti-CD20 H chain vectors
Linear DNA strings encoding for different Fc variants, such as IGHG1*03 or IGHG2*01 allotypes (Fig. 1C), were ordered from Integrated DNA Technologies. DNA strings and pcDNA3.1 expression vectors containing the coding sequences for anti-biotin and anti-CD20 H chain variable regions previously generated from (31)(32)(33), respectively, were digested with HindIII and NheI FastDigest restriction enzymes (Thermo Fisher Scientific) in FastDigest Green Buffer (Thermo Fisher Scientific) at 37˚C for 10 min. The digested H chain vectors were separated on a 1% UltraPure Agarose gel (Thermo Fisher Scientific) with 1:10,000 SYBR Safe (Invitrogen) at 100 V in 13 Tris-acetate-EDTA for 50 min, and the DNA was subsequently purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) according to manufacturer's instructions. Digested DNA strings were purified directly after digestion using the same kit. DNA concentrations were determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).
Ligations were done using a 63 M excess of the insert over the vector with a total amount of 100 ng of DNA in the presence of 400 U of T4 DNA Ligase (New England Biolabs) and 13 T4 DNA Ligase Reaction Buffer (New England Biolabs) in a total volume of 20 ml for 10 min at RT.
Five microliters of the ligation reaction was subsequently added to 50 ml of competent DH5a bacteria (Thermo Fisher Scientific) and incubated on ice for 15 min. Heat shock was performed for 50 s at 42˚C followed by 2 min on ice. Cells were plated on Luria-Bertani (LB) agar plates containing 50 mg/ml ampicillin (Thermo Fisher Scientific) and grown overnight at 37˚C. Single colonies were picked and grown in 2 ml LB-medium containing 50 mg/ml ampicillin (Thermo Fisher Scientific). Precultures were used to inoculate 200 ml LB-medium containing 50 mg/ml ampicillin (Thermo Fisher Scientific) and grown overnight. Plasmids were isolated using a NucleoBond Xtra Maxi Kit (Macherey-Nagel) according to manufacturer's instructions.

Purification of mAbs
Abs were purified from culture supernatants employing an AKTA Prime Plus (GE Healthcare) with a 5-ml Protein G column (GE Healthcare). The ionexchange gradient elution protocol was used with a flow speed of 5 ml/min, applying the low pH elution buffer (0.08 M citric acid/0.04 M Na 2 HPO 4 [pH 3]) over a gradient of 25 ml. One molar Tris-HCL (pH 9) (Thermo Fisher Scientific) was used to neutralize the 1 ml fractions containing the eluted Abs. Fractions were combined and concentrated using 10 kDa m.w. cutoff Protein Concentrator columns (Thermo Fisher Scientific). Subsequently, the Abs were fractionated using high-performance liquid chromatography-size exclusion chromatography using an AKTA UPC-900, P-920, and Frac-950 (GE Healthcare) with a Superdex 200 10/300 GL column (GE Healthcare). Fractions of 0.5 ml were collected at a flow speed of 0.5 ml/min. Monomeric fractions of each Fc variant of the anti-biotin and anti-CD20 Abs were combined and dialyzed overnight at 4˚C to sodium acetate buffer (5 mM [pH 4.5]) or Rituximab buffer (35), respectively. On the next day, the Abs were adjusted to a concentration of 1 mg/ml, aliquoted to working stocks of 20 ml, and subsequently stored at 220˚C. Once thawed, each aliquot was kept at 4˚C and used for a maximum of 1 wk.

SDS-PAGE
Each Ab was tested in SDS-PAGE under reducing and nonreducing conditions. Samples were incubated for 5 min at 70 or 95˚C in the presence of 20 mM iodoacetamide (to prevent minor reduction during denaturation) (36) or 0.25% (w/v) 2-ME in NuPage LDS Sample Buffer (Thermo Fisher Scientific) for reduced and nonreduced conditions, respectively. Samples were loaded on a 7.5% Criterion XT Bis-Tris protein precast gel (Bio-Rad Laboratories) and run with MOPS SDS running buffer (Thermo Fisher Scientific) for 10 min at 100 V followed by 45 min at 120 V. The gel was stained overnight in Blue-Silver solution (10% phosphoric acid, 10% ammonium sulfate, 0.12% Coomassie Blue G-250; Sigma-Aldrich) and 20% methanol (Thermo Fisher Scientific) and thoroughly destained in distilled water.
Separately, biotinylated anti-His mIgG1 (GenScript) was spotted in quadruplicate and 3-fold dilutions, ranging from 30 to 1 nM. Subsequently, 25 nM His-tagged hFcgRIa was loaded onto the sensor before each Ab injection. In this case, Abs were injected at a 3-fold dilution series starting at 4.1 nM until 1000 nM in 13 PBS supplemented with 0.075% (v/v) Tween 80.
Regeneration after every sample was carried out with 10 mM Gly-HCl (pH 2.4) for the hFcgR measurements and 10 mM Gly-HCl (pH 2) for the C1q measurements. In the case of anti-His spots, association and dissociation curves of His-tagged hFcgRIa were subtracted before calculation of IgG binding affinity using SPRINT 1.9.4.4 software (IBIS Technologies). The same was done for the biotinylated BSA spots, where the association and dissociation curves of different anti-biotin Abs were subtracted.
Calculation of the dissociation constant (K D ) was performed by equilibrium fitting to R max = 500, as described earlier (37). Analysis and calculation of all binding data were carried out with Scrubber software version 2 (BioLogic Software) and Excel.

Phagocytosis
For phagocytosis, 1 3 10 7 biotinylated and PKH26-labeled RBCs were opsonized with a 3-fold dilution series of anti-biotin Ab variants starting at a concentration of 3 mg/ml in HEPES buffer in a 96-well V-bottom plate (Corning Costar) for 30 min at RT, shaking at 400 rpm. Afterward, cells were washed 33 with HEPES. The supernatants were discarded, the opsonized RBCs were resuspended in HEPES, and PMNs (FcgRIIa 131H/H-typed) were added to a final volume of 100 ml to reach an E:T ratio of 1:1. The plate was incubated for 45 min at 37˚C, shaking at 175 rpm. Cells were washed once with HEPES, and the remaining RBCs were incubated with lysis buffer for 10 min on ice. After centrifugation, this step was repeated for 5 min, then cells were washed with 13 PBS and measured by FACS. The geometric median fluorescence intensity values for PKH26 were determined for the granulocyte population.

ADCC
A total of 1 3 10 8 biotinylated RBCs were washed twice with 13 PBS, supernatant was discarded, and cells were resuspended in 100 ml of 51 Cr (PerkinElmer) for 45 min at 37˚C at 5% CO 2 . Subsequently, cells were washed twice with 13 PBS and resuspended in IMDM (Life Technologies) containing 10% (v/v) FCS. 51 Cr-labeled RBCs were resuspended in a prepared anti-biotin Ab 5-fold dilution series, starting at a concentration of 0.5 mg/ml (PMNs) or 5 mg/ml (NK cells) in IMDM (Life Technologies) containing 10% (v/v) FCS. For the blocking assays, a 3-fold dilution series of IgG1-Fc wild-type (WT) (evitria) in the same medium was added together with the anti-biotin Abs, starting at a concentration of 167 mg/ml. NK cells or overnight-stimulated neutrophils in IMDM (Life Technologies) with 10% (v/v) FCS were added to opsonized RBCs, reaching a final reaction volume of 100 ml and an E:T ratio of 1:2, and cells were incubated at 37˚C at 5% CO 2 for 2 or 4 h, respectively. Spontaneous and maximum release were accessed by adding IMDM (Life Technologies) with 10% (v/v) FCS or 2.5% saponin (Sigma-Aldrich) (v/v) instead of effector cells, respectively. Percentage of killing was calculated as described previously (12,13).

Fc G236-7 variants have normal integrity and Ag recognition
To investigate the importance of the two glycine residues present at positions 236 and 237, found in human, macaque, and mouse IgG (Fig. 1A), except human IgG2 and mouse IgG1, we generated a set of IgG1 Abs with alanine replacements, deletion of one of the glycine residues (ΔG236), IgG2-WT, and two glycine residues (IgG2+G236) (Fig. 1C). This included the IgG1-G236/7A and the double substitution mutant (GAGA). As a negative control, we combined the well-described Fc-silencing LALA-PG mutations with the N297A substitution (41-43) (Fig. 1B).
After cloning, production, and purification, both anti-CD20 and anti-biotin sets of Abs were tested with high-performance liquid chromatography-size exclusion chromatography, and monomeric fractions were collected. All samples revealed the expected bands in SDS-PAGE under reducing and nonreducing conditions as shown in Supplemental Fig. 1A and 1B. Anti-biotin Abs were confirmed to be functional, as demonstrated by affinity measurements to biotin-BSA in SPR (Supplemental Fig. 2A). Anti-CD20 Abs were confirmed to recognize CD20 on Raji cells by FACS. In line with earlier published data, Ab Fc variants within one subclass were found to bind similarly, whereas IgG2 opsonization levels were generally lower (Supplemental Fig. 2B) (44). With this data, taken together with the analytical data, we concluded that all Abs retained their functionality regarding their Ag recognition as well as overall structural integrity.

FcgR affinity measurements in SPR reveal the importance of G236 for FcgRI binding in human IgG1 and IgG2
To determine how the amino acid substitutions influence the affinities of the mutant Abs to FcgRs, we employed our IBIS SPR streptavidin array equipped with all FcgR with a C-terminal sitespecific biotin tag to guarantee the right orientation. Abs were titrated as analytes, and the K D values were calculated on the basis of equilibrium analysis ( Fig. 2A). WT-IgG1 and -IgG2, as well as the FcgR-silenced IgG1-LALA-N297A-PG, bound to FcgRs as expected (10,12,42,43). IgG1 exhibited strongest binding to FcgRI, followed by FcgRIIIa 158V. IgG2, in contrast, only showed binding to FcgRIIa, with a stronger binding seen for the 131H allotypic variant, as expected (10). The IgG1-LALA-N297A-PG mutant showed no binding to FcgRs (Fig. 2A).
G236A and G237A substitutions in IgG1 did not significantly influence the affinities to FcgRI, but their combination (IgG1-GAGA) showed a significantly lower affinity to FcgRI. IgG1-G236A showed increased binding to both variants of FcgRIIa, which is in line which earlier published data (29). IgG1-G237A and IgG1-GAGA demonstrated no binding to other FcgRs, except for a weak binding to FcgRIIa 131R that was unquantifiable ( Fig. 2A). In contrast, deletion of G236 from IgG1 abrogated binding to all FcgRs (Fig. 2), with only a very weak unquantifiable residual binding to FcgRI ( Fig. 2A). Consistently, we found the reverse; introducing G236 to IgG2 enabled IgG2 binding to FcgRI ( Fig. 2A, 2B). This suggests a direct involvement of G236 in FcgRI interaction, an influence on hinge flexibility, and/or steric hindrance of G236 or adjacent amino acids to be important. Similarly, but to a lesser degree, this mutant also acquired a very weak but detectable binding to FcgRIIIa 158V ( Fig. 2A). At the same time, IgG2 with G236 added lost binding capacity to FcgRIIa, highlighting the unique relationship of IgG2 with FcgR.
DG236, G237A, and GAGA in IgG1 abolish phagocytosis by PMN Next, we investigated the impact of 236/7 variants on ADCC and phagocytosis, using cell types with different FcgR expression profiles to demonstrate the individual effects on FcgRI-, II-, and III-mediated effector functions.
The phagocytosis generally followed the affinity trends for FcgRIIa 131H (IgG1-G236A $ IgG1-WT = IgG2-WT) (Fig. 3C). Also in line with the observed affinity changes, the deletion of G236 from IgG1, as well as the substitutions G237A and GAGA, strongly reduced phagocytosis in our assay (p , 0.001) to a level comparable with IgG1-LALA-N297A-PG ( p , 0.0001). IgG2+G236 showed also a strong reduction in phagocytic capacity but did mediate phagocytosis at a higher concentration, probably because of residual binding to FcgRIIa 131H, inducing increased avidity (13,50) (Fig. 2A). The concentration-dependent shift of the PKH26 signal of the PMN population suggests that next to phagocytosis of whole PKH26-labeled RBCs trogocytosis also occurred (Fig. 3A, Supplemental Fig. 3) (45, 51).

IgG1ΔG236 mediates ADCC via FcgRI only at high concentrations, which is abrogated by irrelevant IgG1-Fc
To investigate the role of FcgRI in PMN effector functions, we stimulated PMNs with IFN-g and G-CSF, resulting in FcgRI upregulation (45,46,52) (Fig. 3B) Of the three IgG1-G237A, IgG1-GAGA, and IgG1ΔG236, incapable of mediating phagocytosis, the first two showed high affinities to FcgRI (Fig. 2). In line with this, these high-affinity variants showed similar PMN-mediated ADCC against IgGopsonized RBC after FcgRI induction. This was only visible for IgG1ΔG236 at high concentrations (Fig. 3D), likely because of the residual affinity to FcgRI.
We then asked whether the residual activity by IgG1ΔG236 is important under slightly more physiological conditions, as irrelevant IgG is present at very high concentration in blood (.10 mg/ml) (53,54). We therefore repeated this experiment with a constant concentration of the IgG variants and increasing amounts of IgG-Fc. Under those conditions the activity of IgG1-WT was constant, but that of IgG1ΔG236 was almost fully abrogated (Fig. 3E).

Increased FcgRIIIa binding of IgG2+G236 translates to CD56 + NK cell ADCC
To investigate the effects of the mutations on FcgRIIIa downstream effector functions, we used NK cells from FcgRIIIa 158V-typed donors and biotinylated RBC as targets. In line with the abrogated affinity of all of the variants to FcgRIIIa, only IgG1-G236A and IgG2+G236 with strong and residual binding to FcgRIIIa, respectively ( Fig. 2A), demonstrated NK cell-mediated ADCC, with IgG2+G236 giving severely weakened effector functions. All other mutants were essentially devoid of FcgRIIIa-mediated effector functions (Fig. 3F).

C1q binding to IgG is differentially affected by G236-7
We also investigated the impact on the interaction with C1q and subsequent complement activation. For this purpose we set up an SPR measurement to determine C1q binding.
The sensorgrams of C1q binding to anti-biotin Abs bound to biotinylated BSA are shown in Fig. 4A. To quantify differences in avidity, a 1:1 binding model was fitted, acknowledging that this does not represent the underlying complexities of the multivalent binding process. The corresponding K D values are plotted in Fig. 4B. The G236A substitution did not have a significant impact on C1q binding, whereas substituting the adjacent G237A resulted in significantly (p , 0.05) lower binding. However, the double GAGA mutations in IgG1 reduced C1q binding strongly (Fig. 4A,  4B). In contrast, no significant changes in affinity for IgG to C1q were observed after either removing G236 from IgG1 or adding it to IgG2 (Fig. 4B). Next, we investigated C1q downstream complement deposition in ELISA-based assays, mimicking the classical complement pathway (7,14,55). C4b and C3b deposition were measured after addition of pooled serum to anti-biotin Abs deposited in biotin-BSA-coated ELISA plates, in a similar manner as described earlier (12). The slight affinity changes measured by SPR resulted in corresponding reduction in binding of C1q to IgG by the ELISA, which were more pronounced than seen by SPR (Fig. 4B, 4C). IgG2-WT and IgG2+G236's ability to mediate complement activation in these assays strictly depended on the Ag density (Supplemental Fig. 4). No C1q binding was seen by IgG1-GAGA (Fig. 4C), nor was it able to trigger C3b and C4b deposition (Fig. 4D, 4E). Interestingly, IgG2+G236 seemed to give elevated complement activity by ELISA as compared with WT IgG2 (Fig. 4C-E).
CDC is unchanged in IgG1-DG236 but is abolished by IgG1-GAGA We then tested the Ab variants for their capacity to mediate CDC. For this we turned to anti-CD20 (rituximab) and CD20 + RAMOS tumor cells. Cells were preopsonized with anti-CD20 Abs and incubated with pooled human serum. IgG1-WT showed  Data shown are a representative of three independent experiments, unless indicated otherwise. All legends have been ranked according to the response at the highest concentration measured. In (C), the response using IgG1-G237A and IgG1-GAGA overlap completely; IgG1DG236 and IgG1-LALA-N297A-PG overlap partially. In (D), the lines of IgG1-G236A, IgG1-G237A, IgG1-GAGA, as well as IgG2-WT and IgG2+G236 largely overlap because of very similar responses. In (E), the lines of IgG1-Fc-WT only and IgG1-LALA-N297A-PG overlap without ADCC. In (F), the lines of IgG1-G237A, IgG1-GAGA, IgG1DG236, IgG2-WT, and IgG1-LALA-N297A-PG largely overlap without ADCC. Statistical analysis was performed using one-way ANOVA and significant differences are indicated with asterisks. ***p , 0.001, ****p , 0.0001. concentration-dependent cell lysis, leading to 100% of dead cells for the highest concentration and having a background of below 20% of dead cells without anti-CD20 Abs (Fig. 5). In contrast to what was expected from the ELISA experiments (Fig. 4C-E), IgG2-WT and IgG2+G236 did not exhibit complement-mediated killing in the cellular CDC assay (Fig. 5B), likely because of differences in epitope density and described subclass-specific differences, leading to lower opsonization levels when deposited on CD20 + cells (Supplemental Fig. 2B) (44). The results for the mutated IgG1 variants were in line with C1q binding and results on complement deposition (Fig. 4): We found no significant differences between IgG1-WT and IgG1-G236A or IgG1-G237A and IgG1ΔG236 (Fig. 5B). IgG1-GAGA showed significantly reduced (p , 0.0001) capability to mediate cell lysis (Fig. 5B). Only at the highest concentration tested did the IgG1-GAGA show a tendency for nonsignificantly elevated CDC (p = 0.6727) compared with IgG1-LALA N297A PG (Fig. 5B), likely explained by residual interaction observed with C1q by SPR (Fig. 4A, 4B).
The relative performance of the Fc variants in comparison with WT-IgG in SPR affinity measurements and cell-based functional assays are summarized in Fig. 6, covering both FcgR-and complement-related results.

Discussion
FcgRs and C1q bind the lower hinge and upper Fc-CH2 region of IgGs. Mutations within this region can influence the interaction of IgGs with FcgRs and/or C1q and thereby also impact their downstream effector functions. Next to understanding the structural and functional importance of single amino acids, function-driven Fc engineering has become a field of interest in the context of Ab-based therapeutics (5,(56)(57)(58)(59).
We recently found that the deletion of G236 from IgG1 (normally absent in germline IgG2) decreased FcRn-dependent placental transport in our model to levels of IgG2, whereas the addition of G236 to IgG2 increased transport to levels of IgG1 (22). We reasoned that G236, given its particular location in the lower hinge region, which is distant from the FcRn-binding site, is an important determinant for Fab flexibility, which may be required for efficient FcRn-mediated transport in the context of vacuolar and, perhaps, tubular transport organelles (22,(60)(61)(62). This is supported by findings from Piche-Nicholas et al. (63), providing evidence that charge distribution in the V region has an impact on FcRndependent recycling caused by close proximity and potential interaction of Fabs and membrane.
As this region is very close to critical FcgR and C1q binding regions, we revisited earlier published data and systematically characterized mutations in position 236 and 237 in the lower hinge region of IgG1 and IgG2. This region has been subjected to mutational analysis in the context of FcgR and/or C1q binding mainly between the 1990s and the early 2000s (16,17,(24)(25)(26)(27)(28).
The binding of C1q to IgG1 has been described to be mainly dependent on D270, K 322, P329, and P331 (15,16). Also, the mutation to alanine of the structurally close G237 has been found to reduce complement activation significantly (17). Substitution of L235 to alanine in IgG1 was reported to substantially reduce C1q binding (17). This suggests A235, which is present in germline IgG2, to be responsible for the comparably low C1q binding of IgG2 (1). Our results add to the knowledge about the IgG:C1q interaction by highlighting the structural importance of the combination of G236 and G237 for C1q binding and downstream complement activation, as neither of the single mutations resulted in a significant reduction; only IgG1-GAGA abrogated complement effector functions. Morgan et al. (17) reported that anti-HLA-DR-IgG1-G237A exhibits reduced CDC activity, which we find to be only slightly reduced. A reason for this could be differing Ag densities and the nature of the epitopes used in the different systems.
IgG2-WT and IgG2+G236 did not exhibit complement-mediated killing in the CDC assay but were able to induce C3b and C4b depositions for ELISA. This is presumably because of a relatively higher Ag density of our hapten than generally found by CD20 on RAMOS cells as well as lower opsonization levels of anti-CD20 Abs on CD20 + cells when formatted to IgG2, confirming earlier published data (44). Curiously, apparently the subclasses diverge qualitatively and quantitatively in this respect, as IgG1-GAGA showed no binding to C1q by ELISAs, whereas residual binding was observed by SPR.
Several groups have investigated the influence of mutations in the lower hinge region on FcgR binding (24,(26)(27)(28). IgG1ΔG236 and IgG2+G236 were previously found not to bind FcgRI (24). Combinations of mutations, among others encompassing ΔG236 in IgG1, were investigated for their impact on FcgRIIa and FcgRIIb interactions (27,28). Our results suggest the crucial importance of G236 for FcgR binding of IgG1 and partially contradict earlier published data. IgG1-G236A has been reported to have increased binding to FcgRIIa (29), which we also found in our SPR binding study. The addition of G236 to IgG2 substantially increased FcgRI and FcgRIIIa binding, and it increased FcgRmediated effector functions accordingly, underlining the importance of G236 for FcgR binding in IgG1 and IgG2. This highlights the evolutionary restrictions of IgG2's interaction with FcgRs in the natural absence of G236, as IgG2 generally only interacts well with FcgRIIa. This interaction is largely lost by insertion of G236. Interestingly, IgG2+G236 also seemed to give elevated complement activity by ELISA. One could speculate that, next to other changes, this suggests that the evolutionarily acquired absence of G236 in IgG2 contributes to its particularly low capacity to activate complement compared with the other IgG subclasses. The deletion of G236 from IgG1 basically abolished binding to all FcgRs, showing no activity in either FcgRIIa-mediated phagocytosis or in FcgRIIIa-mediated NK cell ADCC assays. IgG1ΔG236 only retained residual FcgRI binding in SPR with a more than 100-times reduced affinity but still mediated ADCC with stimulated PMNs at higher concentrations. This is in line with the fact that monomeric interactions measured by SPR do not reflect avidity effects seen for multimeric interactions obtained between effector cells and IgG-opsonized cells (10, 13) or immune complexes (50). Importantly, these FcgRI-mediated effector functions by IgG1ΔG236 were practically abolished in the presence of irrelevant IgG1-Fc, which has much higher affinity for FcgRI. For practical purposes, we only tested irrelevant IgG1-Fcs up to a concentration that was 20 times lower than found for IgGs in serum (53,54), strongly suggesting IgG1ΔG236 to be completely Fc silenced under physiological conditions. Curiously, and despite the crucial importance of G236 in IgG1 for FcgR binding, no significant differences were found for C1q binding or downstream complement activation between IgG1-WT and IgG1ΔG236. This is, to our knowledge, the first single amino acid mutant described with full CDC but abolished FcgRfunctions. Lee et al. (64) published a mutant bearing 2 aa substitutions with the same properties for which they confirmed major conformational changes in the Fc region. As G236 is present in the structural hinge of IgG1 and absent in germline IgG2, we assume its deletion is neither likely to have large impacts on the structure nor strongly promote immunogenicity. It could potentially negatively influence Fab flexibility, however, as IgG2-WT with its shorter hinge has been shown to exhibit a smaller angle between Fabs and Fc region in comparison with IgG1-WT (2). Additionally, the deletion of G236 might influence the steric context of the adjacent amino acids. Mutants, being only capable of mediating complement but not FcgR effector function, are interesting in the context of patients carrying low-FcgR-affinity polymorphisms or in diseases in which FcgR-mediated effector functions are undesired in comparison with complement-mediated clearance (64,65). Enhanced FcgRIII binding by afucosylated Abs has recently been linked to severe disease progression in Ebola and SARS-CoV-2, highlighting the importance of FcgR dead mutations for the development of Ab-based therapies in these diseases . Taken together, our data provide new insights into which amino acids in the lower hinge region of human IgG differentiate FcgR-mediated from complement-mediated effector functions. Furthermore, it could contribute to revealing how IgG2 has evolved to lack FcgRI and C1q binding and therefore have reduced effector functions. The results also show how this region can be tailored to generate Abs with various degrees of FcgR binding with our without the capability to activate complement.