Elimination of Fc Receptor-Dependent Effector Functions of a Modified IgG4 Monoclonal Antibody to Human CD4

Reddy, Manjula; Kinney, Cheryl Ann S.; Chaikin, Margery A.; Payne, Angela; Fishman-Lobell, Jacqueline; Tsui, Ping; Monte, Paul R. Dal; Doyle, Michael L.; Brigham-Burke, Michael; Anderson, Darrell R.; Reff, Mitchell E.; Newman, Roland; Hanna, Nabil; Sweet, Raymond W.; Truneh, Alemseged

doi:10.4049/jimmunol.164.4.1925

Cited by 119 publications

(91 citation statements)

References 40 publications

(45 reference statements)

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“…This, along with our data, suggests that the entire (upper, middle, and lower) hinge in human IgG1 plays a significant role in regulating effector functions. It is likely that this conclusion could be further extended to other isotypes as a mutation in the lower hinge of a primatized IgG4 was shown to modulate ADCC (41). We have shown that the hinge constitutes an attractive target to fine tune the effector functions of a human IgG1.…”

Section: Discussionmentioning

confidence: 91%

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

Dall’Acqua¹,

Cook²,

Damschroder³

et al. 2006

The Journal of Immunology

126

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We report here the engineering of a humanized anti-human EphA2 mAb (mAb 12G3H11) in an effort to explore the relationship between the hinge of a human IgG1 and its effector functions. mAb 12G3H11, used here as a model, is directed against the human receptor tyrosine kinase EphA2, which is an actively investigated target for cancer therapy due to its up-regulation in many cancer cells. Various rational modifications were introduced into the hinge region of mAb 12G3H11. These mutations were predicted to modulate the hinge’s length, flexibility, and/or biochemical properties. We show that the upper and middle hinge both play important, although functionally distinct roles. In particular, middle hinge modifications predicted to decrease its rigidity or length as well as eliminating either one of its two cysteine residues had a strong negative impact on C1q binding and complement-dependent cytotoxicity. Disruption of covalent bonds between both H chains may account in part for these effects. We also describe middle hinge mutants with a significantly decreased ability to bind FcγRIIIA and trigger Ab-dependent cell-mediated cytotoxicity. Conversely, we also generated upper hinge mutants exhibiting an increase in C1q binding and complement-dependent cytotoxicity activity. Therefore, this approach represents a novel strategy to fine-tune the biological activity of a given human IgG1. We also define, for the first time in such a systematic fashion, the relationship between various characteristics of the middle and upper hinge and the corresponding effector functions.

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Section: Discussionmentioning

confidence: 91%

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

Dall’Acqua¹,

Cook²,

Damschroder³

et al. 2006

The Journal of Immunology

126

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“…For further in vivo administration, the Fc regions should preferably be made devoid of effector functions (62)(63)(64)(65) because Fc receptors expressed on peripheral blood cells play an important role in triggering a variety of cytotoxic, phagocytic, and inflammatory functions. The site for binding of IgGs to Fc␥ receptor I (Fc␥RI) is proposed to be Leu-Leu-Gly-Gly-Pro-Ser (EU numbering 234-239), which lies in the lower hinge region (66).…”

Section: Discussionmentioning

confidence: 99%

The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: Implications for peptide vaccination

Rasmussen

Lunde

Michaelsen

et al. 2001

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

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Targeting of antigens to antigen-presenting cells (APCs) increases CD4 ؉ T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110 -120 of hemagglutinin, 323-339 of ovalbumin, and 46 -61 of hen egg lysozyme, were introduced as loops in the C H1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cells in vitro enhances the presentation efficiency by 10 4 to 10 5 compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.O ver recent years, considerable efforts have been directed toward the development of efficient vaccines aimed to stimulate T cells. Effector CD4 ϩ T cells are central in this regard because they regulate B cells and are essential for induction of CD8 ϩ CTL responses through both cytokine secretion (1) and dendritic cell sensitization (2). CD4 ϩ T cells mainly recognize peptides derived from the processing of extracellular antigens by antigen-presenting cells (APCs), presented in association with MHC class II molecules. Targeting of antigen to APCs by coupling to APC-specific Abs increases CD4 ϩ T cell activation, probably because of increased uptake of antigen (3, 4).Several new prospects for preparation of T cell vaccines have been suggested based on the identification and characterization of MHC-associated peptides. Synthetic peptides that correspond to these T cell epitopes may represent ideal subunits for safe vaccines. However, synthetic peptides are poor immunogens because of their small molecular size and very short serum half-life. To circumvent these disadvantages, a variety of recombinant proteins that carry short immunogenic epitopes have been described, and these so-called antigenized molecules represent a new generation of subunit vaccines (5-9). Genetically engineered Ab molecules can function as such delivery systems for T cell peptides (10, 11) and have the advantage of being self-proteins devoid of the side effects sometimes associated with microbial vaccines and, moreover, have a long half-life compared with synthetic peptides.Abs are processed by APCs, and s...

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“…The single amino acid exchange of leucine for glutamic acid at position 235 (EU numbering system) (14) on the border of the hinge and CH2 region has been shown to reduce Fc-mediated effector function through elimination of binding to FcgR (15,29,30). We sought to test the hypothesis that in cocultures, FcFcgR interactions (e.g., involving monocytes) might provide a physiological equivalent to immobilization on plastic, bringing about cross-linking of CD28 or mimicking synapse formation or triggering FcgR-mediated secondary effects that may stimulate PBMCs to secrete cytokines, and explaining the requirement for the Fc region.…”

Section: Responses Of Pbmcs To Nib(28sa)-g4 Nib(28sa)-g1 and Nib(28mentioning

confidence: 99%

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Ball

Fox

Hufton

et al. 2012

The Journal of Immunology

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The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab′)2 and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the “wild-type” IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm–exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab′)2 fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

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Elimination of Fc Receptor-Dependent Effector Functions of a Modified IgG4 Monoclonal Antibody to Human CD4

Cited by 119 publications

References 40 publications

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: Implications for peptide vaccination

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

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