Aglycosylated immunoglobulin G<sub>1</sub>variants productively engage activating Fc receptors

Sazinsky, Stephen L.; Ott, René G.; Silver, Nathaniel W.; Tidor, Bruce; Ravetch, Jeffrey V.; Wittrup, K. Dane

doi:10.1073/pnas.0809257105

Cited by 167 publications

(128 citation statements)

References 36 publications

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“…Also noteworthy is the relatively high affinity of h-13F6 agly for FcγRIII. Aglycosylated IgG is generally considered to be unable (20) or to only very weakly bind FcγRIII (21). Because 13F6 does not neutralize in vitro, the protective activity displayed by h-13F6 agly may be attributable to low-level ADCC activity.…”

Section: Discussionmentioning

confidence: 99%

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Zeitlin

Pettitt

Scully

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

212

195

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No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED 50 = 33 μg). A version with typical heterogenous mammalian glycoforms (ED 50 = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED 50 = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.passive immunization | antibody glycosylation | antibody-dependent cellular cytotoxicity | antiviral

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Section: Discussionmentioning

confidence: 99%

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Zeitlin

Pettitt

Scully

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

212

195

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“…If effector function is desired, compensatory mutations could be made to the C H 2 domain to enable effector function in aglycosylated Fc. 67,68 Furthermore, glycosylated proteins could possibly be www.tandfonline.com 237 mAbs produced by including appropriate enzymes in cell-free reactions. 69 Alternatively, a non-natural amino acid can be incorporated at the glycosylation site N297, 49 followed by site-specific conjugation of glycans in a post-translation manner.…”

Section: Aglycosylated Kih Produced In Xpress Cfmentioning

confidence: 99%

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Lee

Tran

et al. 2015

mAbs

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(2015) Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system , mAbs, 7:1, 231-242, DOI: 10.4161/19420862.2015.989013 To link to this article: https://doi.org/10. 4161/19420862.2015 Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering C H 3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

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“…However, their lack of glycosylation renders them completely unable to bind to FcγRs, and therefore they do not induce FcγR-mediated effector functions (9). Recently, structure-based mutagenesis and, in a separate study screening of small saturation libraries by yeast display, were used to isolate aglycosylated IgG1 variants lacking N297 and carrying mutations in residues S298 and T299 that conferred binding to FcγRIIIa or FcγRIIa, respectively (11,12). However, the respective antibody variants, following expression in HEK293 cells, exhibited significant binding to the inhibitory FcγRIIb receptor (12).…”

mentioning

confidence: 99%

“…Recently, structure-based mutagenesis and, in a separate study screening of small saturation libraries by yeast display, were used to isolate aglycosylated IgG1 variants lacking N297 and carrying mutations in residues S298 and T299 that conferred binding to FcγRIIIa or FcγRIIa, respectively (11,12). However, the respective antibody variants, following expression in HEK293 cells, exhibited significant binding to the inhibitory FcγRIIb receptor (12). This result was not surprising given the high degree of homology between FcγRIIa and FcγRIIb.…”

mentioning

confidence: 99%

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

Jung¹,

Reddy²,

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

144

118

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The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.antibody engineering | bacterial display | bacterial expression | directed evolution | effector function

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Aglycosylated immunoglobulin G₁variants productively engage activating Fc receptors

Abstract: surface plasmon resonance ͉ antibody engineering ͉ directed evolution ͉ yeast display ͉ antibody biomanufacture

Cited by 167 publications

References 36 publications

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

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Aglycosylated immunoglobulin G1variants productively engage activating Fc receptors

Abstract: surface plasmon resonance ͉ antibody engineering ͉ directed evolution ͉ yeast display ͉ antibody biomanufacture

Cited by 167 publications

References 36 publications

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

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Aglycosylated immunoglobulin G₁variants productively engage activating Fc receptors