CD20 is an important target for the treatment of B-cell malignancies,

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. 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 event, 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 draws near are being explored, including new chemotherapies, small molecules, antibody-drug 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 exhibited. In addition, many of these brokers 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 usually 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 Dictamnine supplier (CDC), and antibody-dependent cellular cytotoxicity (ADCC) mediated by cells displaying Fc receptors (FcRs), such as FcRIIIa-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 epitope and/or binding mode have been shown to dictate 2 major types of CD20 antibody effector function profiles, termed type I or type II.5C7 Although both types I and II antibodies bind bivalently to CD20, they form distinct complexes with CD20, as inferred from the fact that the B-cell surface can accommodate approximately double the number of type I antibodies compared with type II. Type I antibodies stabilize CD20 on lipid rafts, leading to stronger C1q binding Rabbit polyclonal to ADAP2 and potent induction of CDC. However, this binding mode triggers only low levels of direct cell death. In contrast, type II antibodies do not stabilize CD20 in lipid rafts and thus exhibit reduced binding to C1q and lower levels of CDC, but they potently induce direct cell death.5 The majority of CD20 antibodies, including rituximab, veltuzumab,8 ocrelizumab,9 and ofatumumab,10 are of type I, whereas the prototype type II antibody is the murine antibody B1 (tositumomab).11 The Fc region of rituximab plays a critical role in triggering the cellular events Dictamnine supplier that lead to B-cell elimination in vivo.7,12,13 This region of the molecule can interact with match protein C1q and FcRs to trigger CDC and ADCC, respectively. Direct cell death mediated by rituximab does not involve the Fc region directly but could potentially be enhanced by Fc-mediated crosslinking via the C1q complex and FcRs.5 Alternative type I CD20 antibodies have been generated, including ofatumumab,14,15 AME-133,16 and a hexavalent anti-CD20 antibody.17 However, superior maximal efficacy over rituximab, that is, efficacy at the saturation point of the dose-response curve, has not been shown for any of these antibodies, and their clinical efficacy compared with rituximab remains to be demonstrated. Our aim was to engineer a novel unconjugated agent against CD20 that displayed enhanced activity. To this end, we report the first Fc-engineered type II CD20 humanized IgG1 antibody, GA101. This manuscript explains Dictamnine supplier the engineering of the variable and Fc regions of GA101 and presents its in vitro and in vivo activity profiles. Methods Antibodies The murine anti-CD20 antibody H299 (W1) was obtained from Beckmann Coulter. Because of its aggregate content, the monomeric fraction was isolated using size exclusion chromatography. Commercial-grade rituximab was obtained from Hoffmann La Roche. GA101 was humanized Dictamnine supplier by grafting the complementarity-determining region sequences from the murine antibody B-ly1 onto the following human frameworks: the VH1-10 plus the JH4 human germline sequences,.

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