ALBERT

Description: GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Description: Background: Treatment of B-cell non-Hodgkin lymphoma (NHL) with antibodies targeting CD20 in conjunction with combination chemotherapy is standard clinical practice. Two different types of CD20 MAb differing significantly in their mode of CD20 binding and biological activities have been identified (Cragg and Glennie. Blood103: 2738–2743, 2004): type I antibodies, as rituximab, are potent in complement mediated cytotoxicity, whereas type II antibodies, as tositumomab, effectively initiate target cell death via caspase-independent apoptosis with concomitant phosphatidylserine exposure. GA101 is a humanized and optimized, third generation, type II CD20 IgG1 antibody that exhibits enhanced ADCC and superior caspase-independent apotosis induction in comparison with currently available CD20 MAbs. Material and Methods: GA101 was humanized by grafting CDR sequences from the murine monoclonal antibody B-ly1 on framework regions with fully human IgG1-kappa germline sequences. During humanization different elbow hinge sequences in the variable region were studied for their capability to induce apoptosis. Furthermore, the Fc region-carbohydrates were glycoengineered using GlycoMAb™ technology leading to bisected, afucosylated Fc region-carbohydrates. Results: The humanized GA101 antibody bound CD20 as type II antibody with nanomolar affinity. Its glycoengineered Fc region bound with 50-fold higher affinity to human FcgammaRIII receptors compared to a standard, non-glycoengineered antibody. Increased FcgammaRIII binding led to a 10–100-fold increase in ADCC against CD20-expressing NHL cell lines. Modification of elbow hinge sequences within the antibody variable framework regions resulted in a strong apoptosis-inducing activity of GA101 upon CD20 binding on target cells. Direct comparison to other CD20 antibodies GA101 showed enhanced apoptosis induction in both a panel of NHL cell lines and ex vivo in samples from patients with a variety of B-cell malignancies. Furthermore, in B-cell depletion assays with whole blood from healthy donors and B-cell leukemic patients, an assay combining ADCC-, CDC- and apoptosis-mediated mechanisms of action, GA101 was significantly more potent and efficacious than other CD20 antibodies, including rituximab and Fc-variants of rituximab that have increased ADCC. Finally, the in vitro superiority of GA101 also translated into superior efficacy in vivo. In NHL xenograft models of different histological origin, including aggressive DLBCL and MCL, treatment with GA101 results in complete tumor remission and long-term survival (cure) compared to tumor stasis, at best, for rituximab. Conclusion: Compared to existing CD20 antibodies GA101 represents a novel, third generation antibody with significantly enhanced efficacy in a variety of in vitro and in vivo preclinical models. GA101 constitutes the first type II CD20 antibody successfully engineered for increased ADCC. Based on these data, GA101 is a promising therapeutic antibody candidate for the treatment of B-cell malignancies.

Description: 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 cell-mediated 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. In nonhuman primates, GA101 demonstrates superior B cell–depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.