Improving Immunotherapy Through Glycodesign

Abstract

Immunotherapy is revolutionizing health care, with the majority of high impact "drugs" approved in the past decade falling into this category of therapy. Despite considerable success, glycosylation-a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeutic-has slowed the development of this class of drugs in the past and remains challenging at present. This article describes how optimizing glycosylation through a variety of glycoengineering strategies provides enticing opportunities to not only avoid past pitfalls, but also to substantially improve immunotherapies including antibodies and recombinant proteins, and cell-based therapies. We cover design principles important for early stage pre-clinical development and also discuss how various glycoengineering strategies can augment the biomanufacturing process to ensure the overall effectiveness of immunotherapeutics.

Keywords: antibody-dependent cell cytotoxicity (ADCC); antibody-drug conjugates (ADCs); glycoengineering; glycosylation; immunotherapy; metabolic glycoengineering; monoclonal antibodies.

Figure 1

Overview of Biologics with immunotherapy-related examples. (A) “Biologics” is a broad term that refers to any therapy created using material derived from a living system, several examples are shown [as adapted from Chhina (8)]. (B) Protein-based biologics dominate today's commercial products with examples discussed in this article including monoclonal antibodies (section Antibodies) and interferon (section Blocking Antibodies). (C) Until a few decades ago, vaccines dominated immunotherapy, a 200-year old endeavor (section Vaccines), with cancer vaccines (section O-Glycans in Immunotherapy and 3.3) representing one example of this trend today. (D). The extraordinarily diverse nature of immunotherapy is illustrated by emerging cell-based (e.g., CAR T-cell, section Chimeric Antigen Receptor (CAR) T-cell Therapy) and gene therapies.

Figure 2

Branch elongation and structural diversity of N-glycans. The Glc₃Man₉GlcNAc₂-P-P-dolichol LLO structure is synthesized in the ER where it is further processed and transferred to the Golgi resulting in high mannose (e.g., Man₅GlcNAc₂), hybrid, and complex type N-glycans that undergo branching via Mgat1, 2, 4, and 5 GlcNAc transferase activity that respectively creates di-, tri-, or tetra-antennary structures. Following the initial branching step, the glycan structure may be fucosylated or undergo additional elongation and capping modifications (Top panel). Alternatively, Mgat3 may add a bisecting GlcNAc residue which blocks Mgat4 and 5 activity thereby preventing tri- and tetra-antennary and further terminal diversification (bottom). The presence of a bisecting GlcNAc also hinders core fucosylation (red triangle) and reduces the capacity for downstream elongation and capping. [All glycan symbol structures in this figure and throughout this document were made using software from Cheng and coauthors (30)].

Figure 3

The role of N-linked glycosylation in mAb function and other aspects of immunity. (A) IgG type antibodies have two N-linked glycosylation sites at Asn297 of the Fc region that usually bear biantennary complex type N-glycans elongated with zero (G0), one (G1), or two (G2) galactose residues. (B) The presence of fucose and sialic acid inhibits FcγRIIIa binding resulting in lower ADCC activity; conversely, the anti-inflammatory character of sialic acid makes its presence desirable for IVIG therapy. (C) The presence (or absence) of sialic acid affects binding to the ASGP receptor, resulting in quick recycling of asialylated therapeutic proteins, which reduces serum half-life. By contrast, sialylation block ASGP receptor-mediated recycling, improving pharmacokinetic properties. (D) Neu5Ac added to galactose in an α2,3-linkage elicits a certain set of biological responses, one of which is—as part of the sLe^x epitope (shown in Figure 4)—to facilitate immune cell trafficking throughout the body by enabling “tethering and rolling” steps of leukocyte extravasation from the vascular system. (E) Neu5Ac in an α2,6-linkage elicits a distinct set of response, including binding to Siglec receptors (45), where in the example shown, adapted from Büll et al. (46), this moiety modulates macrophage activity. (F) Core fucose, in particular in the α1,6-linkage, inhibits ADCC requiring higher mAb antibodies compared to defucosylation drug [adapted from GlycoWord (47)]. Glycans can also result in unwanted immunogenicity ranging from mild, chronic responses emanating from Neu5Gc (G) (48), to life-threatening, anaphylactic responses from α-Gal (H) (49).

Figure 4

Structural diversity of mucin-type O-glycans. Mucin type O-glycan biosynthesis begins with the transfer of GalNAc to serine or threonine. The GalNAc monosaccharaide can be left unmodified but is typically extended to create eight different core structures that can be further modified with single monosaccharides, Lewis structural epitopes, blood group antigen groups, or other glycan epitopes (e.g., the cancer-related sT or sTn antigens).

Figure 5

O-Glycans in normal and cancerous MUC1 and MUC1-based cancer vaccine development. (A) The MUC1 protein core (green) is composed of a 20 amino acid tandem repeat with each unit having five potential O-glycosylation sites. (B) MUC1 is overexpressed in numerous cancers (not shown) and is characterized by truncated O-glycans (shown). (C) MGE can be used to introduce non-natural chemical moieties (e.g., Sia5Prop and Sia5PhAc) to enhance the immunogenicity of tumor-associated cancer antigens (TACAs). As shown in the inset (bottom), antibodies can be developed to the glycoengineered TACAs and used to immunize a tumor-bearing animal (D). (E) Supplementation with the MGE analog induces expression of the non-natural version of the TACA, resulting in tumor-selective binding and stimulation of the immune system to recognize and eradicate the tumor (F).

Figure 6

Structure of lipopolysaccharide (LPS). (A) Glycolipids, exemplified by bacterial structures such as LPS contain the Lipid A, and inner core, an outer core, and the O-antigen, which varies based on species and strain [Salmonella enterica Serotype Typhi is show (160)]. (B) LPS glycans contains a variety of non-mammalian monosaccharides, which contributes to their immunogenicity and provokes sepsis [(A,B) are adapted from Saeui et al. (161)]. (C) Medicinal chemistry efforts have exploited the Lipid A structure to create anti-inflammatory analogs [three are shown, from Piazza et al. (162)] that are promising anti-sepsis agents.

Figure 7

Glycosphingolipids (GSL) structures and role in immunotherapy. (A) Human GSLs are derived from ceramide upon addition of galactose (to form “GalCer”) or, more commonly, addition of glucose (to form “GlcCer”); a fraction of GlcCer is further elaborated with galactose to form “LacCer,” which is the building block for lacto(neo)series, globosides, and gangliosides as cataloged elsewhere (21); here [in (B)] we show several GSLs currently targeted by immunotherapy.

Figure 8

Glycoengineering mAbs for enhanced sialylation and glycan-targeted ADC production. (A) Cells can be supplemented with ManNAc or analogs (e.g., Ac₄ManNAc or 1,3,4-O-Bu₃ManNAc), which intercept and increase flux through the sialic acid biosynthetic pathway with the indicated relative efficiencies (“R.E.” values) increasing sialylation of recombinant glycoproteins, such as mAbs. (B) Alternatively, cells can be supplemented with analogs containing non-natural chemical moieties (e.g., Ac₄ManNAz or 1,3,4-O-Bu₃ManNAz to install azide groups or Ac₄6-Thio-Fuc to install thiols). These functional groups, which do not naturally occur in glycans, constitute chemical handles for conjugation to small molecules including drugs, toxins, or imaging agents.