Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell–mediated B-cell cytotoxicity

Abstract

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 ...

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“… 101 Chimeric IgG1     Obinutuzumab/ GA101CD20Reduced (<30%)Coexpression with GnT III and α-ManII in CHO cells (GlycoMAb Technology)Enhanced tumor inhibition of GA101 compared with rituximab in human lymphoma xenograft mouse modelsMossner et al. 102 Humanized IgG1   Enhanced B-cell depletion in spleen and lymph nodes of cynomolgus monkeys over rituximabDalle et al. 131     Enhanced tumor growth inhibition over rituximab in human-transformed follicular lymphoma RL model in SCID mice. RituximabCD20afucosylatedCommercial rituximab treated with endoglycosidase/fucosidase to generate GlcNAc-rituximabEnhanced depletion of huCD20 + B cells in an FcγR-humanized mouse model over original rituximabLi et al.…”

“…Reduced fucosylation is achieved through the co-expression of GnT-III and αManII in CHO cells. The antibody demonstrated an enhanced binding affinity for FcγRIIIa and consequently, an increased ADCC activity 102 . Results from Phase1b/2 trials indicated that all patients with CLL experienced rapid and sustained removal of B cells in the peripheral blood.…”

Preclinical Studies on the Mechanism of Action and the Anti-Lymphoma Activity of the Novel Anti-CD20 Antibody GA101

“…Depending on their ability to redistribute CD20 into lipid rafts and to induce CDC, or to induce cell death and homotypic adhesion, anti-CD20 mAb can be classified as Type I or II CD20 antibodies. Schematically, Type I antibodies (such as rituximab) induce CDC with redistribution of CD20 into lipid rafts, whereas type II mAB (such as the murine antibody tositumomab, or the humanized antibody GA101) are believed to act primarily through the direct induction of nonclassical cell death and exhibit low CDC activity (29).…”

B-Cell Depletion with CD20 Antibodies as New Approach in the Treatment of Inflammatory and Immunological Events Associated with Spinal Cord Injury

“…The dose of 18B12 (30 mg/kg) used here was based on that used in a previous study [23]; the dose-response and timecourse studies were previously done at our laboratory (data not shown).…”

“…Because it has been demonstrated that anti-CD20 treatment eliminates pre-existing B-cell activity in human autoimmunity [21], the purpose of the current proposal has been to evaluate the effects of treatment with glycoengineered anti-CD20 antibody 18B12 in mice subjected to SCI [22], in an attempt to investigate the anti-inflammatory and immunological potential of this anti-CD20 drug. 18B12 has been glycoengineered to make a nonfucosylated Fc region that substantially enhances the ability to attach and recruit effector cells, and hence to an increased antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell phagocytosis compared with nonglycoengineered fully fucosylated antibodies [23,24].…”