scholarly journals Site-specific conjugation of native antibody

2020 ◽  
Vol 3 (4) ◽  
pp. 271-284
Author(s):  
Amissi Sadiki ◽  
Shefali R Vaidya ◽  
Mina Abdollahi ◽  
Gunjan Bhardwaj ◽  
Michael E Dolan ◽  
...  
Keyword(s):  
Selective Reduction ◽  
Stochastic Methods ◽  
Site Specificity ◽  
Specific Chemical ◽  
Site Specific ◽  
Drug Conjugates ◽  
Low Concentrations ◽  
Antibody Conjugates ◽  
Dose Dependent ◽  
Complementarity Determining Region

ABSTRACT Traditionally, non-specific chemical conjugations, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity-determining region may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein, we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody–drug conjugates in clinical trials along with the future perspectives of these site-specific methods.

scholarly journals Antibody conjugation and formulation

2019 ◽  
Vol 2 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Nathan J Alves
Keyword(s):  
Oral Delivery ◽  
Clinical Implementation ◽  
High Concentration ◽  
Site Specific ◽  
Routes Of Administration ◽  
Drug Conjugates ◽  
Large Size ◽  
Low Concentrations ◽  
Antibody Conjugates ◽  
Antibody Conjugation

ABSTRACT In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market, the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates, more accessible routes of administration such as intramuscular and subcutaneous are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of ADCs and the demands of formulation for high concentration clinical implementation.

scholarly journals Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches

2021 ◽  
Vol 14 (4) ◽  
pp. 343
Author(s):  
Ahmad Fawzi Hussain ◽  
Armin Grimm ◽  
Wenjie Sheng ◽  
Chaoyu Zhang ◽  
Marwah Al-Rawe ◽  
...  
Keyword(s):  
Therapeutic Agents ◽  
Amino Acid Residues ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Site Specific ◽  
Drug Conjugates ◽  
Complement Dependent Cytotoxicity ◽  
Antibody Conjugates ◽  
Specific Antigens ◽  
Antibody Conjugate ◽  
Therapeutic Mechanisms

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.

scholarly journals Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex (hGQ) DNAzyme Nanostructures

2020 ◽  
Author(s):  
Jordi Keijzer ◽  
Bauke Albada
Keyword(s):  
Heavy Chain ◽  
Protein Modification ◽  
Site Specificity ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Synthetic Dna ◽  
Drug Conjugates ◽  
Sds Page ◽  
G Quadruplex ◽  
Antibody Drug

<div>Synthetic DNA that forms various G-quadruplex nanostructures, in combination with hemin, <i>N</i>-methyl luminol derivatives, and H2O2 can site-specifically modify proteins (i.e. evidence is provided for lysozyme and human alpha-thrombin). The catalytic modification is completed in 15-30 mins, and the site-specificity is influenced by the G-quadruplex topology (a total of 22 G-quadruplex forming sequences was tested). We also show that the heavy chain of the therapeutic antibody trastuzumab is modified, which facilitates the preparation of antibody-drug conjugates. Furthermore, a trigger can be programmed into this synthetic DNA so that the protein modification chemistry is made dependent on an external trigger.</div><div><br></div>Techniques used: HPLC, SDS-PAGE, LC-MS/MS, NMR.

A novel site-specific chemical conjugation of IgG antibodies by affinity peptide for immunoassays

2020 ◽  
Author(s):  
Satoka Mori ◽  
Arisa Abe ◽  
Naoto Ishikawa ◽  
Abdur Rafique ◽  
Yuji Ito
Keyword(s):  
Protein A ◽  
Disease Diagnosis ◽  
Conjugation System ◽  
Specific Chemical ◽  
Site Specific ◽  
Drug Conjugates ◽  
Chemical Conjugation ◽  
Affinity Peptide ◽  
A Site ◽  
Human And Mouse

Abstract Recently, there has been an increasing interest in site-specific modifications of antibodies used in immunoassays for disease diagnosis and as antibody therapeutics, such as antibody−drug conjugates. Previously, we established a site-specific chemical conjugation system using an IgG-Fc binding chemical conjugation affinity peptide (CCAP). CCAP could be used only for the modification of human IgG owing to the lack of affinity of CCAP to rodent IgG molecules. In this study, novel CCAP reagents are proposed, which can be used for both human and mouse IgG, based on the Staphylococcus aureus protein A domain-derived affinity peptides Z34C and Z33. Compared with the activity of a conventional randomly modified antibody, mouse IgG modified using this method had favourable features in two immunoassays, demonstrating the advantages of the proposed CCAP method in preserving antibody functionality during conjugation.

scholarly journals Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex (hGQ) DNAzyme Nanostructures

2020 ◽  
Author(s):  
Jordi Keijzer ◽  
Bauke Albada
Keyword(s):  
Heavy Chain ◽  
Protein Modification ◽  
Site Specificity ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Synthetic Dna ◽  
Drug Conjugates ◽  
Sds Page ◽  
G Quadruplex ◽  
Antibody Drug

<div>Synthetic DNA that forms various G-quadruplex nanostructures, in combination with hemin, <i>N</i>-methyl luminol derivatives, and H2O2 can site-specifically modify proteins (i.e. evidence is provided for lysozyme and human alpha-thrombin). The catalytic modification is completed in 15-30 mins, and the site-specificity is influenced by the G-quadruplex topology (a total of 22 G-quadruplex forming sequences was tested). We also show that the heavy chain of the therapeutic antibody trastuzumab is modified, which facilitates the preparation of antibody-drug conjugates. Furthermore, a trigger can be programmed into this synthetic DNA so that the protein modification chemistry is made dependent on an external trigger.</div><div><br></div>Techniques used: HPLC, SDS-PAGE, LC-MS/MS, NMR.

scholarly journals A Platform for the Generation of Site-Specific Antibody–Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines

2020 ◽  
Vol 31 (9) ◽  
pp. 2136-2146
Author(s):  
Ruud G. E. Coumans ◽  
Gerry J. A. Ariaans ◽  
Henri J. Spijker ◽  
Pascal Renart Verkerk ◽  
Patrick H. Beusker ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Selective Reduction ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

scholarly journals Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems

2021 ◽  
pp. 175-190
Author(s):  
Marlitt Stech ◽  
Nathanaël Rakotoarinoro ◽  
Tamara Teichmann ◽  
Anne Zemella ◽  
Lena Thoring ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Eukaryotic Cell ◽  
Fluorescent Dye ◽  
Site Specific ◽  
Drug Conjugates ◽  
Sds Page ◽  
Antibody Conjugates ◽  
Cell Free Protein Synthesis ◽  
Antibody Drug

AbstractCell-free protein synthesis (CFPS) enables the development of antibody conjugates, such as fluorophore conjugates and antibody-drug conjugates (ADCs), in a rapid and straightforward manner. In the first part, we describe the cell-free synthesis of antibodies containing fluorescent non-canonical amino acids (ncaa) by using pre-charged tRNA. In the second part, we describe the cell-free synthesis of antibodies containing ncaa by using an orthogonal system, followed by the site-specific conjugation of the fluorescent dye DyLight 650-phosphine. The expression of the antibodies containing ncaa was analyzed by SDS-PAGE, followed by autoradiography and the labeling by in-gel fluorescence. Two different fluorescently labeled antibodies could be generated.

Reactive Nanopattern in a Triple Structured Bio-Inspired Honeycomb Film as a Clickable Platform

2018 ◽  
Author(s):  
Pierre Marcasuzaa ◽  
Samuel Pearson ◽  
Karell Bosson ◽  
Laurence Pessoni ◽  
Jean-Charles Dupin ◽  
...  
Keyword(s):  
Self Assembly ◽  
Double Porosity ◽  
Surface Groups ◽  
Specific Chemical ◽  
Site Specific ◽  
Surface Functionality ◽  
Wenzel State ◽  
Breath Figure ◽  
Responsive Surfaces ◽  
Secondary Population

A hierarchically structured platform was obtained from spontaneous self-assembly of a poly(styrene)-<i>b</i>-poly(vinylbenzylchloride) (PS-<i>b</i>-PVBC) block copolymer (BCP) during breath figure (BF) templating. The BF process using a water/ethanol atmosphere gave a unique double porosity in which hexagonally arranged micron-sized pores were encircled by a secondary population of smaller, nano-sized pores. A third level of structuration was simultaneously introduced between the pores by directed BCP self-assembly to form out-of-the-plane nano-cylinders, offering very rapid bottom-up access to a film with unprecedented triple structure which could be used as a reactive platform for introducing further surface functionality. The surface nano-domains of VBC were exploited as reactive nano-patterns for site-specific chemical functionalization by firstly substituting the exposed chlorine moiety with azide, then “clicking” an alkyne by copper (I) catalyzed azide-alkyne Huisgen cycloaddition (CuAAC). Successful chemical modification was verified by NMR spectroscopy, FTIR spectroscopy, and XPS, with retention of the micro- and nanostructuration confirmed by SEM and AFM respectively. Protonation of the cyclotriazole surface groups triggered a switch in macroscopic behavior from a Cassie-Baxter state to a Wenzel state, highlighting the possibility of producing responsive surfaces with hierarchical structure.

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