Best Niche Antibody-based Biologics

Regarding Aglycosylated Antibody Therapeutics

by Yen-Ming Hsu

03/31/2023

Immunoglobulin G (IgG) is the humoral arm of the adaptive immune system. An individual IgG molecule is made of two heterodimers each consists of one heavy chain and one light chain joined by the disulfide linkages at the hinge region. In each heavy chain, there is a single glycan attached to the Asn 297 (N297) residue near the top of the CH2 region. This N-linked glycan is critical for the structure and functions of the IgG. IgG has two functional activities endowing with it the ability to serve as a liaison between the pathogens and the immune effector functions. The effector functions include the cellular (Fc receptor-expressing leukocytes) and soluble (complement) components. Human IgG Fc receptors include the FcγR1, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB and FcRn. FcγRI binds to monomeric IgG Fc with high affinity. In contrast, the monomeric IgG does not bind to FcγRII and FcγRIII unless it is bound with multi-valent antigens wherein secure binding of the immunocomplexes to these Fc receptors with avidity interaction. FcRn transports the IgG across the placenta. It also protects IgG from being proteolyzed while cycling through the endocytic vesicles. Stable binding to FcRn under mild acidic conditions in these vesicles is essential for IgG’s long half-life in circulation.

With the dual functional activities, Immunoglobulin can bind to an antigen on target cells through the Fab region, followed by engaging Fc receptors expressed on the surface of immune cells through the Fc region, and triggering antibody-dependent cell mediated cytotoxicity (ADCC), or antibody-dependent cell mediated phagocytosis (ADCP), or to recruit serum complement and trigger complement-mediated cytotoxicity (CDC).

Bindings of Fcγ receptors and C1q are N-297-linked glycan dependent

Asn-297 linked glycan at the upper CH2 region governs the conformation of the overlapped binding sites of complement C1q and Fcγ receptors. Removal of the N-linked glycan leads to less constrained conformation at the upper CH2 region resulting in lowered thermal stability and compromised interaction with Fcγ receptor and lead to inability to trigger ADCC, ADCP and CDC. FcRn binds to the juncture between CH2 and CH3 regions. Removal of the N-297-linked glycan does not affect antibody’s interaction with FcRn. Hence, normal IgG and its aglycosylated counterpart exhibit similar half-life in circulation.

Structurally, the presence of the N-297-linked glycan stabilizes the conformation around the joined upper CH2 regions and confers the resistance to thermal denaturation and aggregation caused by low pH conditions. Functionally, the N-297-linked glycan is essential for IgG to interact with all Fc receptors with the exception of FcRn. In the absence of this glycan, IgG is no longer capable of performing its “liaison” function resulting loss of its ability to trigger ADCC, ADCP and CDC. FcRn binds to the juncture of heavy chain CH2 and CH3 regions. Thus, its binding is not affected by removal of N-297 glycan as such normal IgG and its aglycosylated counterpart exhibit a comparable half-life in circulation.

For oncological applications, the effector functions of the therapeutic antibody are highly desirable. On the other hand, for many other applications where the MOA (mechanism of action) of therapeutic antibody is meant to be neutralizing, agonistic, or antagonistic, the effector functions is not necessary or may even be undesirable. As a therapeutical candidate glycosylated IgG has some advantages in developmental process despite its slightly altered biophysical properties. One advantage is a simpler manufacturing process as the heterogeneity of the oligosaccharides associated with the N297 glycan is no longer a concern. In addition, the aglycosylated IgG can be produced more economically using the bacteria and yeast. Examples of the aglycosylated IgG as clinical candidate including TRX1, TRX4, and TRX 518 from Tolerx (Cambrdige, Massachusetts, USA), ALD518 from Alder Biopharmaceuticals (Bothell, Washington, USA), and OA-5D5 from Roche (San Francisco, California, USA) indicating that aglycosylated IgGs are suitable for therapeutical applications. 

Choosing a control antibody is critical for testing the specific activity of an antibody of interest. AB Biosciences has engineered a series of bona fide control antibodies (Please see Z-MAB®) including all isotypes of human, mouse and rat origins. Z-MAB® has no active antigen-binding activity yet retains the overall antibody structure and conformation. It is therefore an excellent control agent for all antibody studies. To generate the matched aglycosylated control antibody, AB Biosciences introduced the N297A mutation to each of the wildtype ZmAbs. Hence for each isotype IgG of human, mouse and rat origins, ZmAbs with wildtype Fc and the aglycosylated Fc are available (Z-MAB®). Specifically the aglycosylated Z-mAb offers an unprecedented advantage for evaluting the specific activity of aglycosylated antibodies of interest.

 

References 

  1. Dmitrij Hristodorov, Rainer Fischer, Lars Linden (2013) With or without sugar? (A)glycosylation of therapeutic antibodies. Mol Biotechnol 54:1056-68.
  2. Man-Seok Ju and Sang Taek Jung (2014) Aglycosylatedfull-length IgG antibodies: steps toward next-generation immunotherapeutics. Curr Opin Biotechnol. 30:128-39.
  3. Yusuke Mimura, Toshihiko Katoh, Radka Saldova, Roisin O'Flaherty, Tomonori Izumi, Yuka Mimura-Kimura, Toshiaki Utsunomiya, Yoichi Mizukami, Kenji Yamamoto, Tsuneo Matsumoto, Pauline M Rudd  (2018) Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety, functionality and efficacy. Protein Cell 9:47-62.
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