Novel 3rd Generation Humanized Type II CD20 Antibody with Glycoengineered Fc and Modified Elbow Hinge for Enhanced ADCC and Superior Apoptosis Induction.

Abstract

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.

Development of Novel Anti-Cd20 Monoclonal Antibodies and Modulation in Cd20 Levels on Cell Surface: Looking to Improve Immunotherapy Response

“…Recently (in November 2013) it has FDA approved mAb for use in combination with chlorambucil to treat patients with previously untreated chronic lymphocytic leukemia (CLL). Preclinical studies suggested that the modified Fc region of GA101 improved about 50 fold binding affinity to FcγRIII and 10 to 100 fold increased cell death through ADCC mechanism against CD20 positive NHL cell lines [ 57 – 60 ]. Moreover, in vitro study also demonstrated that modification in elbow hinge regions promotes direct programmed cell death mechanism in several NHL cell lines and primary malignant B-cells [ 12 , 49 ].…”

Radiolabeled and Native Antibodies and the Prospect of Cure of Follicular Lymphoma

“…Other antibodies directed against different target proteins in lymphoma and leukemia are under investigation in preclinical and clinical trials. Further improved biological efficacy in NHL might be achieved by combining anti-CD20 rituximab treatment with other antibodies directed against different antigens, such as anti-CD22 [34] or anti-CD40 [35], using humanized antibodies, or using novel anti-CD20 antibodies with modified Fc domains that provide greater affinity for Fc receptors and superior effector functions [36,37].…”

Treatment of chronic lymphoid leukemias with monoclonal antibodies: current place and perspectives

“…GA-101 (Hoffman La Roche) is a novel third generation fully humanized and optimized anti-CD20 IgG1 differing significantly from other anti-CD20 MAbs, such as rituximab [Fries et al, 2007;Umana et al, 2006]. Using GlycoMab technology, the Fcregion of GA-101 was glycoengineered to contain bisected, afucosylated carbohydrates.…”