CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cellmediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. IntroductionRituximab, a type I chimeric IgG1 anti-CD20 antibody, has revolutionized the management and treatment of B-cell malignancies, increasing the median overall survival of patients with many of these diseases. 1 In combination with chemotherapy, it has significantly improved response rates and progression-free and overall survival of patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 1,2 Rituximab treatment has also benefited patients with other diseases amenable to B-cell depletion therapy, including B-cell chronic lymphocytic leukemia (B-CLL) and rheumatoid arthritis. 2,3 Nevertheless, relapse is a common occurrence, for example, in B-CLL, and there remains a need for treatments that delay the onset of relapse without increasing toxicity. 1 To this end, various therapeutic approaches are being explored, including new chemotherapies, small molecules, antibodydrug conjugates, and the use of alternative B-cell targets. However, in contrast to the situation with rituximab, the clinical benefit of these therapies remains to be demonstrated. In addition, many of these agents exhibit poor safety and tolerability profiles or necessitate the use of more complex treatment regimens.Thus far, CD20 has been the most effective unconjugated antibody target for the treatment of B-cell malignancies. An alternative and complementary approach is to generate new unconjugated CD20 antibodies with enhanced functional activities that may lead to superior efficacy. Three types of functional activities of anti-CD20 antibodies have been described: signaling in target cells on CD20 binding leading to growth inhibition and (nonclassic) apoptosis (referred to as "direct cell death"), complement-dependent cytotoxicity (CDC), and antibodydependent cellular cytotoxicity (ADCC) mediated by cells displaying Fc␥ receptors (Fc␥Rs), such as Fc␥RIIIa-expressing NK cells and macrophages. 4,5 Anti-CD20 antibodies with different functions may be generated either (1) by selecting antibodies that bind to a different CD20 epitope, which bind in an alternative mode or with changed affinity, resulting in altered intensity or type of functional mechanism; or (2) by engineering the Fc region of the antibody to enhance immune effector functions. The ...

Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydratecarbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.immunoglobulin | afucosylation | antibody effector function | X-ray crystallography

The immune response depends on the binding of opsonized antigens to cellular Fc receptors and the subsequent initiation of various cellular effector functions of the immune system. Here we describe the crystal structures of a soluble Fc gamma receptor (sFc gammaRIII, CD16), an Fc fragment from human IgG1 (hFc1) and their complex. In the 1:1 complex the receptor binds to the two halves of the Fc fragment in contact with residues of the C gamma2 domains and the hinge region. Upon complex formation the angle between the two sFc gammaRIII domains increases significantly and the Fc fragment opens asymmetrically. The high degree of amino acid conservation between sFc gammaRIII and other Fc receptors, and similarly between hFc1 and related immunoglobulins, suggest similar structures and modes of association. Thus the described structure is a model for immune complex recognition and helps to explain the vastly differing affinities of other Fc gammaR-IgG complexes and the Fc epsilonRI alpha-IgE complex.

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Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

Immunoglobulins recognize and clear microbial pathogens and toxins through the coupling of variable region specificity to Fc-triggered cellular activation. These proinflammatory activities are regulated, thus avoiding the pathogenic sequelae of uncontrolled inflammation by modulating the composition of the Fc-linked glycan. Upon sialylation, the affinities for Fcγ receptors are reduced, whereas those for alternative cellular receptors, such as dendritic cellspecific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23, are increased. We demonstrate that sialylation induces significant structural alterations in the Cγ2 domain and propose a model that explains the observed changes in ligand specificity and biological activity. By analogy to related complexes formed by IgE and its evolutionarily related Fc receptors, we conclude that this mechanism is general for the modulation of antibody-triggered immune responses, characterized by a shift between an "open" activating conformation and a "closed" antiinflammatory state of antibody Fc fragments. This common mechanism has been targeted by pathogens to avoid host defense and offers targets for therapeutic intervention in allergic and autoimmune disorders.conformational change | sialylated IgG Fc I gG and IgE mediate their proinflammatory properties through the crosslinking of the 1:1 complex of the Fc receptor (FcR) monomer in the Fc dimer cleft (1, 2). By contrast, both IgG and IgE can engage a second class of receptors, the evolutionarily related, C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (3) and CD23 (4), respectively, resulting in anti-inflammatory and immunosuppressive responses (5, 6). The structural basis for the ability of IgE to interact either with one or the other of these two disparate classes of receptors has recently been defined (7). The intrinsic flexibility of the IgE Ce3 domain results in both open and closed conformations of the IgE Fc, resulting in the binding of either FceRI or CD23, respectively. Binding of either receptor induces an allosteric change in the IgE Fc to the alternative conformation, thus precluding the interaction with the other receptor (7). Binding of IgE to the type II, C-type lectin CD23 is neither carbohydrate-nor calcium-dependent, mediated exclusively through protein-protein interactions, generating a 2:1 complex of CD23 with the Ce3-Ce4 interface (7). DC-SIGN is a structurally homologous, calcium-dependent, carbohydratebinding, type II lectin, tightly linked to CD23 on chromosome 19 (8), displaying ligand specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens. Binding of DC-SIGN to IgG requires that the complex, biantennary glycan, attached to the evolutionarily conserved glycosylation site Asn-297 and enclosed within the cavity formed by the Cγ2 domains of the A and B chains of the Fc dimer, be processed to the α2,6 sialylated form (9, 10). Importantly, no evidence has been found for DC-SIGN binding to sialylate...

Engagement of Fc␥ receptors (Fc␥Rs; ⌬H, ؊6.5 kcal mol ؊1 ; T⌬S, 1.9 kcal mol ؊1 ; ⌬C p , ؊160 cal mol ؊1 K ؊1 ). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C H 2 domains but only slightly to sFc␥RIIb binding. Truncation of 1,3-and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic ⌬H, and a more negative ⌬C p for sFc␥RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C H 2 domains abrogated sFc␥RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C H 2 domains, impairing sFc␥RIIb binding.

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-Å resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.Streptococcus pyogenes ͉ Mac-1