AllergoOncology: Expression platform development and functional profiling of an anti‐HER2 IgE antibody

Ilieva, Kristina M.; Fazekas-Singer, Judit; Bax, Heather J.; Crescioli, Silvia; Montero-Morales, Laura; Mele, Silvia; Sow, Heng Sheng; Stavraka, Chara; Josephs, Debra H.; Spicer, James; Steinkellner, Herta; Jensen‐Jarolim, Erika; Tutt, Andrew; Karagiannis, Sophia N.

doi:10.1111/all.13818

Cited by 12 publications

(31 citation statements)

References 9 publications

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“…Using N. benthamiana and applying extensive glycoengineering, we generated IgE variants with identical protein backbones but distinct N -glycosylation patterns that mainly differ in their core glycosylation and terminal residues, i.e., N -acetylglucosamine (GlcNAc), galactose or sialic acid. In addition, HER2-IgE was produced in human embryonic kidney (Expi293F) cells (Ilieva et al, 2019) and, as observed in our previous investigation (Montero-Morales et al, 2017), exhibited a mixture of 30 glycoforms (most abundantly, core-fucosylated branched glycans terminating with galactose or sialic acid). Using ELISA and cell-based assays, we show that all recombinant IgE variants bind to the HER2 antigen and to the high affinity FcεRI, irrespectively of their glycosylation pattern.…”

Section: Introductionsupporting

confidence: 55%

“…OST has been used previously to increase NGS occupancies of recombinant human proteins expressed in plants (Castilho et al, 2018). HER2-IgE was also produced in the Expi293™ Expression System (Thermo Fischer Scientific), as recently described (Ilieva et al, 2019).…”

Section: Resultsmentioning

confidence: 99%

“…Cloning and expression of HER2-IgE in HEK cells (Expi293F™ expression system, HER2-IgE HEK ) were described earlier (Ilieva et al, 2019). The cloning and expression of the isotype control chimeric NIP-IgE HEK , against the hapten 5-iodo-4-hydroxy-3-nitrophenyl, were described earlier (Neuberger et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

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In Planta Glycan Engineering and Functional Activities of IgE Antibodies

Montero-Morales

Maresch

Crescioli

et al. 2019

Front. Bioeng. Biotechnol.

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Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.

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Section: Introductionsupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

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In Planta Glycan Engineering and Functional Activities of IgE Antibodies

Montero-Morales

Maresch

Crescioli

et al. 2019

Front. Bioeng. Biotechnol.

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“…This observation is consistent with our previous findings showing maximal activation of human macrophages by IgE stimulation even in the absence of FcεR saturation [ 22 ]. Cross-linking of a proportion of IgE-FcεRI complexes on the cell surface has been shown to be sufficient for maximum mast cell and basophil activation and mediator release [ 45 , 46 , 47 ]. Monocytes already have FcεRs partly occupied by endogenous IgE and thus exogenous provision of MOv18 IgE will result in a fraction of FcεRs bearing tumour-specific IgE.…”

Section: Discussionmentioning

confidence: 99%

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype

Nakamura

Souri

Osborn

et al. 2020

Cancers

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IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

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“…Taken together, these attributes make IgE a molecule with much potential to target cancer. Therefore, pipelines to rapidly and cost-effectively clone, express and purify IgE antibodies for pre-clinical study have been developed [ 32 , 35 , 36 , 37 , 38 , 39 ] and indeed, in the field of AllergoOncology [ 25 , 26 , 34 , 40 , 41 ], in vitro and in vivo data (described below) have demonstrated the promising therapeutic efficacy of tumour-targeting IgE antibodies.…”

Section: Introductionmentioning

confidence: 99%

IgE Antibodies against Cancer: Efficacy and Safety

et al. 2020

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Immunoglobulin E (IgE) antibodies are well known for their role in allergic diseases and for contributions to antiparasitic immune responses. Properties of this antibody class that mediate powerful effector functions may be redirected for the treatment of solid tumours. This has led to the rise of a new class of therapeutic antibodies to complement the armamentarium of approved tumour targeting antibodies, which to date are all IgG class. The perceived risk of type I hypersensitivity reactions following administration of IgE has necessitated particular consideration in the development of these therapeutic agents. Here, we bring together the properties of IgE antibodies pivotal to the hypothesis for superior antitumour activity compared to IgG, observations of in vitro and in vivo efficacy and mechanisms of action, and a focus on the safety considerations for this novel class of therapeutic agent. These include in vitro studies of potential hypersensitivity, selection of and observations from appropriate in vivo animal models and possible implications of the high degree of glycosylation of IgE. We also discuss the use of ex vivo predictive and monitoring clinical tools, as well as the risk mitigation steps employed in, and the preliminary outcomes from, the first-in-human clinical trial of a candidate anticancer IgE therapeutic.

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AllergoOncology: Expression platform development and functional profiling of an anti‐HER2 IgE antibody

Cited by 12 publications

References 9 publications

In Planta Glycan Engineering and Functional Activities of IgE Antibodies

In Planta Glycan Engineering and Functional Activities of IgE Antibodies

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype

IgE Antibodies against Cancer: Efficacy and Safety

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