scholarly journals Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations

Antibodies ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 22
Author(s):  
Souad Boune ◽  
Peisheng Hu ◽  
Alan L. Epstein ◽  
Leslie A. Khawli
Keyword(s):  
Modern Medicine ◽  
Sialic Acids ◽  
Igg Subclass ◽  
Post Translational Modifications ◽  
Effector Functions ◽  
Terminal Galactose ◽  
Complement Dependent Cytotoxicity ◽  
Dependent Cell ◽  
Ch2 Domain ◽  
The Stability

The development of recombinant therapeutic proteins has been a major revolution in modern medicine. Therapeutic-based monoclonal antibodies (mAbs) are growing rapidly, providing a potential class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Most mAbs are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. Post-translational modifications here account for the observed high heterogeneity of glycoforms that may or not impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Moreover, mAbs produced in non-human cells express oligosaccharides that are not normally found in serum IgGs might lead to immunogenicity issues when administered to patients. This review summarizes our understanding of the terminal sugar residues, such as mannose, sialic acids, fucose, or galactose, which influence therapeutic mAbs either positively or negatively in this regard. This review also discusses mannosylation, which has significant undesirable effects on the PK of glycoproteins, causing a decreased mAbs’ half-life. Moreover, terminal galactose residues can enhance CDC activities and Fc–C1q interactions, and core fucose can decrease ADCC and Fc–FcγRs binding. To optimize the therapeutic use of mAbs, glycoengineering strategies are used to reduce glyco-heterogeneity of mAbs, increase their safety profile, and improve the therapeutic efficacy of these important reagents.

scholarly journals A common glycan structure on immunoglobulin G for enhancement of effector functions

2015 ◽  
Vol 112 (34) ◽  
pp. 10611-10616 ◽  
Author(s):  
Chin-Wei Lin ◽  
Ming-Hung Tsai ◽  
Shiou-Ting Li ◽  
Tsung-I Tsai ◽  
Kuo-Ching Chu ◽  
...  
Keyword(s):  
Immune Cells ◽  
Immunoglobulin G ◽  
Therapeutic Agents ◽  
Binding Activity ◽  
Sialic Acids ◽  
Glycan Structure ◽  
Effector Functions ◽  
Complement Dependent Cytotoxicity ◽  
Infection And Inflammation ◽  
Dependent Cell

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

scholarly journals Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

2021 ◽  
Vol 12 ◽  
Author(s):  
Adrian Elter ◽  
Desislava Yanakieva ◽  
David Fiebig ◽  
Kerstin Hallstein ◽  
Stefan Becker ◽  
...  
Keyword(s):  
Therapeutic Antibodies ◽  
Antibody Isotype ◽  
Effector Functions ◽  
Antibody Therapeutics ◽  
Drug Conjugates ◽  
Complement Dependent Cytotoxicity ◽  
Fold Reduction ◽  
New Strategy ◽  
Protease Activation ◽  
Dependent Cell

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

scholarly journals Enhancing CDC and ADCC of CD19 Antibodies by Combining Fc Protein-Engineering with Fc Glyco-Engineering

Antibodies ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 63
Author(s):  
Sophia Roßkopf ◽  
Klara Marie Eichholz ◽  
Dorothee Winterberg ◽  
Katarina Julia Diemer ◽  
Sebastian Lutz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Engineering ◽  
Amino Acid Sequence ◽  
Therapeutic Antibodies ◽  
Adcc Activity ◽  
Fcγ Receptor ◽  
Effector Functions ◽  
Complement Dependent Cytotoxicity ◽  
Dependent Cell ◽  
Cluster Of Differentiation

Background: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody’s Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies. Results: Four versions of a CD19 antibody based on tafasitamab’s V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently. Conclusions: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

scholarly journals Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

2016 ◽  
Vol 113 (42) ◽  
pp. 11931-11936 ◽  
Author(s):  
Wenqian He ◽  
Gene S. Tan ◽  
Caitlin E. Mullarkey ◽  
Amanda J. Lee ◽  
Mannie Man Wai Lam ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Critical Role ◽  
Host Cells ◽  
Viral Glycoprotein ◽  
Effector Functions ◽  
Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

Unique Toxicities and Resistance Mechanisms Associated with Monoclonal Antibody Therapy

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 329-334 ◽  
Author(s):  
Jonathan W. Friedberg
Keyword(s):  
Follicular Lymphoma ◽  
Single Agent ◽  
Antibody Therapy ◽  
Resistance Mechanisms ◽  
Complement Dependent Cytotoxicity ◽  
Dependent Cell ◽  
Impact On Treatment ◽  
Anti Cd20 ◽  
In Vivo Cytotoxicity

Abstract Anti-CD20 therapy has had a truly dramatic impact on treatment and outcome of patients with follicular lymphoma. Unfortunately, the majority of responses to single-agent rituximab are incomplete, and all patients with follicular lymphoma will experience disease progression at some point following rituximab therapy. Rituximab has multiple mechanisms of inducing in vivo cytotoxicity, including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, direct apoptotic signaling, and possible vaccinal effects. The cellular microenvironment within follicular lymphoma has a profound impact on which mechanism is dominant, and confers resistance in many situations. Both tumor-associated and host-associated factors also contribute to rituximab resistance. There are multiple potential approaches to overcoming rituximab resistance, including rational biologic combination immunotherapy, engineered antibodies, and radioimmunoconjugates. Improved ability to overcome resistance will require further elucidation of critical signaling pathways involved in rituximab induced cytotoxicity and a comprehensive understanding of interactions between its multiple mechanisms of action.

Abstract 42: Differential Contribution Of The N- And C-terminal Domains To The Folding And Function Of Tandem Calponin-homology Domains

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Krishna Mallela ◽  
Swati Bandi ◽  
Surinder Singh ◽  
Geoffrey Armstrong
Keyword(s):  
Full Length ◽  
Actin Binding ◽  
Main Function ◽  
Calponin Homology ◽  
Terminal Domain ◽  
Binding Domains ◽  
Binding Function ◽  
Ch2 Domain ◽  
The Stability ◽  
And Function

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability. This work was funded by the AHA Grant 11SDG4880046.

scholarly journals The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto’s Thyroiditis: An in Vitro Model of Thyroid Autoimmunity

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 171 ◽  
Author(s):  
Marta Ząbczyńska ◽  
Katarzyna Polak ◽  
Kamila Kozłowska ◽  
Grzegorz Sokołowski ◽  
Ewa Pocheć
Keyword(s):  
Cell Line ◽  
Hashimoto’S Thyroiditis ◽  
Leukemia Cell Line ◽  
Hashimoto's Thyroiditis ◽  
Target Cells ◽  
Healthy Donors ◽  
Complement Dependent Cytotoxicity ◽  
Dependent Cell ◽  
Igg Glycosylation

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto’s thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.

scholarly journals Engineering IgG-Like Bispecific Antibodies—An Overview

Antibodies ◽  
2018 ◽  
Vol 7 (3) ◽  
pp. 28 ◽  
Author(s):  
Simon Krah ◽  
Harald Kolmar ◽  
Stefan Becker ◽  
Stefan Zielonka
Keyword(s):  
Lymphoblastic Leukemia ◽  
Treatment Options ◽  
Malignant Ascites ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Clotting Factors ◽  
Effector Functions ◽  
Antibody Therapeutics ◽  
Complement Dependent Cytotoxicity ◽  
Therapeutic Concepts

Monoclonal antibody therapeutics have proven to be successful treatment options for patients, which have various indications. Particularly in oncology, therapeutic concepts involving antibodies often rely on the so-called effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are programed in the antibody Fc region. However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells. Furthermore, long term treatments can lead to resistance against the applied drug, which is monospecific by nature. There is promise in using specific antibodies to overcome such issues due to their capability of recruiting and activating T-cells directly at the tumor site, for instance. During the last decade, two of these entities, which are referred to as Blinatumomab and Catumaxomab, have been approved to treat patients with acute lymphoblastic leukemia and malignant ascites. In addition, Emicizumab, which is a bispecific antibody targeting clotting factors IXa and X, was recently granted market approval by the FDA in 2017 for the treatment of hemophilia A. However, the generation of these next generation therapeutics is challenging and requires tremendous engineering efforts as two distinct paratopes need to be combined from two different heavy and light chains. This mini review summarizes technologies, which enable the generation of antibodies with dual specificities.

scholarly journals Studies on the synthesis and stability of α-ketoacyl peptides

Amino Acids ◽  
2020 ◽  
Vol 52 (10) ◽  
pp. 1425-1438
Author(s):  
Johann Sajapin ◽  
Michael Hellwig
Keyword(s):  
Peptide Bond ◽  
Side Chain ◽  
Peptide Backbone ◽  
Post Translational Modifications ◽  
Degradation Reactions ◽  
Keto Acids ◽  
Strecker Aldehydes ◽  
The Stability ◽  
Dehydration Processes ◽  
Hydrolysis Of

Abstract Oxidative stress, an excess of reactive oxygen species (ROS), may lead to oxidative post-translational modifications of proteins resulting in the cleavage of the peptide backbone, known as α-amidation, and formation of fragments such as peptide amides and α-ketoacyl peptides (α-KaP). In this study, we first compared different approaches for the synthesis of different model α-KaP and then investigated their stability compared to the corresponding unmodified peptides. The stability of peptides was studied at room temperature or at temperatures relevant for food processing (100 °C for cooking and 150 °C as a simulation of roasting) in water, in 1% (m/v) acetic acid or as the dry substance (to simulate the thermal treatment of dehydration processes) by HPLC analysis. Oxidation of peptides by 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) proved to be the most suited method for synthesis of α-KaPs. The acyl side chain of the carbonyl-terminal α-keto acid has a crucial impact on the stability of α-KaPs. This carbonyl group has a catalytic effect on the hydrolysis of the neighboring peptide bond, leading to the release of α-keto acids. Unmodified peptides were significantly more stable than the corresponding α-KaPs. The possibility of further degradation reactions was shown by the formation of Schiff bases from glyoxylic or pyruvic acids with glycine and proven through detection of transamination products and Strecker aldehydes of α-keto acids by HPLC–MS/MS. We propose here a mechanism for the decomposition of α-ketoacyl peptides.

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