T. Shantha Raju

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement‐dependent cytotoxicity (CDC) and antibody‐dependent (Ab‐dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non‐glycosylated IgGs (Biochem. Biophys. Res.Commun. 2006, 341, 797–803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), β‐galactose (G2), or N‐acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix‐assisted laser desorption‐time‐of‐flight‐mass spectroscopy (MALDI‐TOF‐MS) and high‐performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform‐dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.

Diversity in structure and functions of antibody sialylation in the Fc

Terminal sialic acid residues of glycoconjugates exhibit remarkable functional and structural diversity. They affect biological activity, serum half-life and structural stability of glycoproteins. Alternatively, they act as mediators for pathogens to invade host systems. These surface exposed N-glycans are easily accessible for interactions with receptors, enzymes, etc. In contrast, Fc N-glycans of IgGs are sequestered within the two CH2 domains and exhibit high degree of heterogeneity. They are required for antibody effector functions including binding to C1q protein. Biological significance of Fc glycans has been extensively studied and importance of terminal galactose, bisecting GlcNAc and core fucose has been realized. This review focuses on the recent advances in structure and functions of terminal sialic acid residues of Fc glycans.

Avidity confers FcγR binding and immune effector function to aglycosylated immunoglobulin G1.

Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc‐mediated immune effector functions. Human IgGs have an N‐glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N‐linked glycosylation site has been reported to “silence” FcγR‐binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen‐engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes. Copyright

Solubility evaluation of murine hybridoma antibodies

The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.

Assessing Fc Glycan Heterogeneity of Therapeutic Recombinant Monoclonal Antibodies Using NP-HPLC

Recombinant monoclonal antibodies (rMAbs) are becoming major human therapeutics to treat life-threatening diseases such as cancer. These rMAbs are produced using either in vitro cell culture processes or transgenic technology in animals or plants. Glycans present in the Fc region can affect functions of rMAbs. These Fc glycans are heterogeneous and impact binding of rMAbs to Fc gamma receptors (FcγRs) and C1q protein. As a result Fc glycans affect antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of rMAbs. Thus understanding the glycan heterogeneity is necessary during the development of these rMAbs as human therapeutics. Because of their biological significance, understanding the glycan structure and their impact on the function of antibody molecules is also a regulatory requirement. Glycan mapping by NP-HPLC with fluorescence detection is a sensitive and reproducible method. Labeling of released glycans with anthranilic acid (AA) using reductive amination procedure improves sensitivity of detection. The NP-HPLC method resolves both neutral and sialylated glycans, thus enabling the user to obtain a broad heterogeneity profile of Fc glycans in a single run. Added advantage of the method is that the labeled glycans can be characterized using mass spectrometry and the method is also amenable for LC-MS analysis.

A peptide immunization approach to counteract a Staphylococcus aureus protease defense against host immunity

Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.

Glyco-engineering of Fc Glycans to Enhance the Biological Functions of Therapeutic IgGs

Glycans N-linked to the Fc region of IgGs affect binding to various Fc receptors and C1q protein and therefore are important for IgG effector functions, including ADCC and CDC activities. Fc glycans are highly heterogeneous and the nature of this variation differs between species. Heterogeneity of Fc glycans arises from the presence or the absence of different terminal sugars, including sialic acid, galactose and N-acetylglucosamine, core fucose along with bisecting N-acetylglucosamine. To understand the influence of individual terminal sugar residues on serum half-life and antibody effector functions, it is necessary to prepare homogeneous IgG glycoforms. This chapter describes glycoengineering strategies to prepare IgG molecules containing homogeneous glycan chains in the Fc region and their significance in assessing IgG functions. The importance of selecting appropriate in vitro and/or in vivo conditions, including enzymes, buffers and cell culture conditions, to produce recombinant IgGs with homogeneous glycoforms is discussed.

Abstract 3518: Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies

The unique ability of flow cytometry to simultaneously examine intricate details of multiple cell subsets is unparalleled and the platform is now a dominant tool for measuring antibody mediated effector functions on cancer cells. The objective of this study is to illustrate the utility of flow cytometry as a single platform for concurrently measuring the multiple mechanisms of actions of therapeutic antibodies such as ADCC, ADCP, Apoptosis, CDC and Trogocytosis on human cancer cells and immune functioning cells. Rituximab® was tested with the Burkett9s Lymphoma cell line (Daudi) for all assays between the ranges of 0.01 and 100ug/mL. Apoptosis was evaluated by measuring Caspase 3/7 staining. ADCC assays were established with Rituximab®/CFSE labeled Daudi cells and human PBMC’s. ADCP was assessed with Rituximab®/ CFSE labeled Daudi cells and CD14+/CD11b+ in vitro generated phagocytes using a 4 hour exposure. CDC assays were established with Daudi cells/ 10% pooled human serum using 7-AAD as the cytotoxic indicator in a 4 hour assay. Trogocytosis was measured up to 4 hours with Rituximab®/ Daudi and PBMC9s and CD19/20 and CD14. All assays were analyzed using a FACS ARIA III flow cytometer and replicates of 3 were evaluated for statistical relevance. Results demonstrated that Rituximab® induced effects were observed in all 5 assays using flow cytometry. Apoptosis was induced when 0.01ug/mL Rituximab® was present. ADCP occurred at levels as low as 0.01ug/mL Rituximab®. ADCC and CDC occurred at levels as low as 0.1ug/mL Rituximab®. Trogocytosis, indicated by the transfer of CD19+ B cells to CD14+ monocytes, occurred at 0.1ug/ml Rituximab®. In summary, we demonstrated the clinical applicability of flow cytometry as a single platform to simultaneously evaluate five different mechanisms of actions that can occur when therapeutic antibodies are used to treat cancer cells. Citation Format: Antony R. Chadderton, Shane Harvey, Brandy Strake, Jill Giles-Komar, Renold J. Capocasale, T. Shantha Raju. Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3518.