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

Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc -portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. These oligosaccharide moieties introduce a higher degree of heterogeneity within the molecule, by influencing stability of the antibody and its mediated effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). The carbohydrate moieties can vary strongly depending on the production host and can be manipulated by different fermentation conditions, thereby influencing the function of the antibody. Therefore it is necessary to carefully monitor changes in the carbohydrate composition during cell line development and production processes. This chapter describes two different mass spectrometry based methods used for analyses of the carbohydrate moieties attached to the Fc-part of human IgG1. In the first approach, the glycans are released from the antibody by endoglycosidase (Peptide N Glycosidase F) digestion and monitored by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS), whereas in the second method the carbohydrate structures, still attached to an enzymatically produced Fc-fragment, are analyzed by electrospray ionization mass spectrometry.

MCSP/CSPG4 is a large transmembrane proteoglycan identified in melanomas as HMW-MAA. In the mouse it is known as neurite growth factor 2 (NG2), a marker of pericyte recruitment. MCSP has been used as a target for clinical imaging of (uveal) melanomas by immunoscintigraphy. MCSP shows uniform and abundant expression in ca. 60-80% of melanoma, and was described in lobular breast carcinoma, glioblastoma, osteo- & chondrosarcoma, and basal cell carcinoma. It is present at high levels on pericytes of tumor neovasculature, but down-regulated as vessels mature. Normal tissue expression is low and it is not detected on PBMCs. We have generated human/Cynomolgus cross-reactive antibodies against a membrane-proximal MCSP epitope by mouse immunization with a linear peptide derived from the membrane proximal D3 domain followed by boosting with melanoma cells. The mouse antibody LC007 was selected for humanization due to its potent induction of ADCC as a chimeric antibody, compared to antibodies to membrane distal epitopes of MCSP. LC007 as chimeric IgG1 and its humanized IgG1 derivative M4-3-ML2 are characterized by the following properties: i) Specific binding to the native epitope on MCSP+ melanoma cells, but no induction of internalization; ii) Specific IHC staining of MCSP+ cells in FFPET samples; iii) ca 10 nM monovalent affinity for hMCSP D3 domain. Moreover, glycoengineering of LC007 and M4-3-ML2 antibodies using GlycoMab technology resulted in increased binding affinity for hFcgRIIIa and enhanced ADCC potency and absolute killing of melanoma cell lines. As expected, neither up to 10 ug/mL wildtype, nor glycoengineered M4-3-ML2 induced relevant cytokine (IL-6, TNF-α, IFN-γ) release in human whole blood supporting that MCSP is not expressed there. Subsequently, we studied anti-tumoral efficacy of the chimeric antibody LC007 and the humanized antibody M4-3-ML2 in disseminated models of MV3 and MDA-MB435 melanoma after i.v. injection of tumor cells in hCD16 transgenic Scid mice, which express the functional human high affinity FcgRIIIa receptor on NK cells. Both, glycoengineered LC007 and M4-3-ML2 mediated efficacy in terms of enhanced median and overall survival in both disseminated xenograft models, and were superior to the respective non-glycoengineered antibodies. Taken together, our studies support MCSP/CSPG4 as an attractive target for antibody-based cancer immunotherapy. Further studies investigating the anti-angiogenic effect of MCSP antibodies via their action on pericytes/vascular smooth muscle cells are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-236. doi:1538-7445.AM2012-LB-236

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