Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.     

Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin's B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.     

IgG2m4, an engineered antibody isotype with reduced Fc function

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fcγ receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.     

Biological activity in the human system of isotype variants of oligosaccharide-Y-specific murine monoclonal antibodies

Summary The capacity of isotype variants of BR55-2, an anti-tumor monoclonal antibody directed against Y oligosaccharide, to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the human system was evaluated using freshly isolated peripheral blood mononuclear cells, lymphocytes, monocytes and complement. The ADCC activities of the BR55-2 IgG3 isotype and its switch variants (IgG1, IgG2b, and IgG2a) with human monocytes were high for all isotypes, whereas the activity of all isotypes was lower with freshly isolated lymphocytes, IgG1 being the least effective. The CDC on the other hand was strong with IgG3, IgG2b and IgG2a and negative with the IgG1 variant. The IgG3 and IgG2a isotypes were selected for further development. Their strong ADCC and CDC activity against mammary carcinoma, colon carcinoma and small-cell lung tumor cell lines was confirmed quantitatively using proteins highly purified from tissue-culture supernatants. Significant variations in ADCC and CDC competence was observed among human donors.This work was supported by grants CA 10 815, CA 25 874 and CA 21124 from the National Institutes of Health     

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.     

Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.     

A matched set of rat/mouse chimeric antibodies. Identification and biological properties of rat H chain constant regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha

Rat C regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha have been characterized by means of chimeric antibody technology. A set of rat/mouse Ag-specific (anti-4-hydroxy-3-nitrophenacetyl) antibodies was constructed that differ only in the H chain constant region but carry identical V region and L chain, both of which are of mouse origin. All rat constant regions could be expressed and m.w. were as expected from the protein sequence. A slight variation in mobility within the IgG subclasses allowed us to establish a hierarchy for the sizes of the four gamma H chains; gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. Rat IgG2c and IgG2b could be purified on both protein A and protein G while rat IgG2a could only be purified on protein G. Rat IgM and IgG2b were the most potent in C-mediated hemolysis. This was not simply a consequence of the amount of C1q bound because IgG2c bound C1q efficiently but was relatively poor in cell lysis. In ADCC using human effector and target cells, IgG2b and IgG1 were the most effective.     

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.Key words: antibody, Fc, complement, CDC, C1q, ADCC, phagocytosis, CD20, CD19, CD40     

Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcγRIIIa receptor binding activity of an IgG1 antibody

N‐linked Fc glycosylation of IgG1 monoclonal antibody therapeutics can directly influence their mechanism of action by impacting IgG effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). Therefore, identification and detailed characterization of Fc glycan critical quality attributes (CQAs) provides important information for process design and control. A two‐step approach was used to identify and characterize the Fc glycan CQAs for an IgG1 Mab with effector function. First, single factor experiments were performed to identify glycan critical quality attributes that influence ADCC and CDC activities. Next, a full‐factorial design of experiment (DOE) to characterize the possible interactions and relative effect of these three glycan species on ADCC, CDC, and FcγRIIIa binding was employed. Additionally, the DOE data were used to develop models to predict ADCC, CDC, and FcγRIIIa binding of a given configuration of the three glycan species for this IgG1 molecule. The results demonstrate that for ADCC, afuco mono/bi has the largest effect, followed by HM and β‐gal, while FcγRIIIa binding is affected by afuco mono/bi and β‐gal. CDC, in contrast, is affected by β‐gal only. This type of glycan characterization and modeling can provide valuable information for development, manufacturing support and process improvements for IgG products that require effector function for efficacy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1181–1192, 2016     

Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off.

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.     

Exogenous C1q reconstitutes resident but not inflammatory mouse peritoneal macrophages for Fc receptor-dependent cellular cytotoxicity and phagocytosis. Relationship to endogenous C1q availability

Murine resident peritoneal macrophages (PM) were refractory to activation for antibody-dependent cellular cytotoxicity (ADCC) of SRBC targets as compared with either oil or thioglycollate-elicited inflammatory macrophages. Western blot analysis of macrophage cellular lysates indicated a direct correlation between the endogenous C1q levels and their innate response to activation for ADCC. Inflammatory PM had 7- to 14-fold higher C1q levels (ca. 23 to 45 ng C1q/100 micrograms protein) than resident PM (ca. 3 ng C1q/100 micrograms protein) as determined by densitometric scanning of blots. Purified exogenous mouse or human C1q were found to reconstitute the response of resident PM for ADCC mediated by C-activating mouse IgG2a or IgG2b mAb, but not by non-C-activating IgG1. Thioglycollate-elicited PM with highest endogenous C1q levels were unaffected by exogenous C1q, whereas oil-elicited PM with intermediate C1q levels were slightly augmented in their ADCC response by exogenous C1q. Augmentation of the resident PM response for ADCC activation was accomplished by either coincubation of effector macrophages with physiologic concentrations of C1q (0.5 to 4.0 micrograms/ml), IgG, and SRBC targets or by IgG and C1q preopsonized targets. FcR-dependent phagocytosis by resident PM was similarly reconstituted by exogenous C1q. The results indicate that resident macrophages with low potential for C1q biosynthesis and secretion were reconstituted by exogenous C1q in their FcR-dependent phagocytosis and ADCC, whereas inflammatory macrophages with sufficient endogenous C1q levels were largely unaffected. Thus C1q appears to have a pivotal mechanistic role in the initiation of macrophage activation for FcR-dependent effector functions.     

Cytolytic activity of murine anti-dog lymphoma monoclonal antibodies with canine effector cells and complement

Peripheral blood leukocytes (PBL), nonadherent lymphocytes, and adherent monocytes separated from freshly isolated blood of 15 dogs were analyzed for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in combination with murine anti-tumor monoclonal antibodies (MAbs). Canine monocytes isolated from most donors by adherence to gelatin-fibronectin-coated plastic surface presented high ADCC activity against the canine lymphoma 17-71 tumor cell line in combination with antilymphoma MAbs 231 (IgG2a) and 234-2a (IgG2a). Canine lymphocytes generally showed lower ADCC activity than total PBL or monocytes. Canine PBL effector cells showed high ADCC activity against the human colorectal carcinoma SW948 cell line using the Y-6-specific MAb isotype switch variants 55-2 IgG3, 55-2 IgG1, 55-2 IgG2b, and 55-2 IgG2a. Analysis of the role of murine MAb isotypes on ADCC activity against tumors by canine cells using anti-human tumor class-switch variant MAbs and a panel of anti-canine lymphoma MAbs of different IgG subclass revealed the highest ADCC activity with MAbs of the IgG2a and IgG3 subclasses. IgG2a antilymphoma MAbs were also able to lyse tumor cells in complement-dependent cytotoxicity (CDC) assay. These results suggest the potential value of MAbs of IgG3 and IgG2a subclasses in immunotherapy against canine lymphoma.     

An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum

Among nonneutralizing HIV-1 envelope antibodies (Abs), those capable of mediating antibody-dependent cellular cytotoxicity (ADCC) activity have been postulated to be important for control of HIV-1 infection. ADCC-mediating Ab must recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fcγ receptor (FcR) of the effector cell population. However, the precise targets of serum ADCC antibody are poorly characterized. The human monoclonal antibody (MAb) A32 is a nonneutralizing antibody isolated from an HIV-1 chronically infected person. We investigated the ability of MAb A32 to recognize HIV-1 envelope expressed on the surface of CD4(+) T cells infected with primary and laboratory-adapted strains of HIV-1, as well as its ability to mediate ADCC activity. The MAb A32 epitope was expressed on the surface of HIV-1-infected CD4(+) T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.     

RV144 HIV-1 vaccination impacts post-infection antibody responses

The RV144 vaccine efficacy clinical trial showed a reduction in HIV-1 infections by 31%. Vaccine efficacy was associated with stronger binding antibody responses to the HIV Envelope (Env) V1V2 region, with decreased efficacy as responses wane. High levels of Ab-dependent cellular cytotoxicity (ADCC) together with low plasma levels of Env-specific IgA also correlated with decreased infection risk. We investigated whether B cell priming from RV144 vaccination impacted functional antibody responses to HIV-1 following infection. Antibody responses were assessed in 37 vaccine and 63 placebo recipients at 6, 12, and 36 months following HIV diagnosis. The magnitude, specificity, dynamics, subclass recognition and distribution of the binding antibody response following infection were different in RV144 vaccine recipients compared to placebo recipients. Vaccine recipients demonstrated increased IgG1 binding specifically to V1V2, as well as increased IgG2 and IgG4 but decreased IgG3 to HIV-1 Env. No difference in IgA binding to HIV-1 Env was detected between the vaccine and placebo recipients following infection. RV144 vaccination limited the development of broadly neutralizing antibodies post-infection, but enhanced Fc-mediated effector functions indicating B cell priming by RV144 vaccination impacted downstream antibody function. However, these functional responses were not associated with clinical markers of disease progression. These data reveal that RV144 vaccination primed B cells towards specific binding and functional antibody responses following HIV-1 infection.     

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.     

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild‐type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.     

Interaction of monocytes with tumor cells coated with complement with or without antibody

Raji, a human B lymphoblastoid cell line has the ability to activate the complement cascade by alternate pathway mechanisms with subsequent fixation of C3 to receptors on the Raji cell membrane. Using this property, we examined the role that complement plays in mediating a cytolytic event between human peripheral blood monocytes and Raji cells coated with C3b, antibody, or both. Presence of C3 was confirmed by immune adherence. IgG bound to the Raji membrane was quantitated using I¹²⁵ Staphylococcal protein A assay. The presence of alternate pathway-activated C3 on Raji cells failed to produce monocyte-mediated cytotoxicity. These same target cells subsequently coated with antibody concentration ranging from 200 to >600,000 SPA molecules per Raji cell produced neither enhancement nor inhibition of antibody-dependent, cell-mediated cytotoxicity (ADCC). ADCC was enhanced by complement when complement activation and binding of C3 to the cell surface occurred by classical pathway mechanisms. ADCC of 32% ± 3.2 occurred with undiluted antiserum (625,000 SPA molecules bound/Raji cell) with enhancement to 52% ± 1.1 in the presence of C3. IgG inhibition of ADCC was unaffected by the presence of membrane-bound C3.     

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.     

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

Background

Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.

Methods

We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.

Results

ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.

Conclusion

A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.