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 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 Bioorthogonal Prodrug–Antibody Conjugates for On-Target and On-Demand Chemotherapy

CCS Chemistry ◽  
2019 ◽  
Vol 1 (2) ◽  
pp. 226-236 ◽  
Author(s):  
Feng Lin ◽  
Long Chen ◽  
Heng Zhang ◽  
William Shu Ching Ngai ◽  
Xiangmei Zeng ◽  
...  
Keyword(s):  
Side Effects ◽  
Anticancer Agent ◽  
Tissue Penetration ◽  
Tumor Penetration ◽  
On Demand ◽  
Drug Conjugates ◽  
Antibody Conjugates ◽  
Antibody Conjugate ◽  
Chemotherapy Agents ◽  
Premature Release

Current antibody–drug conjugates (ADCs) suffer from low tissue penetration and significant side effects, largely due to the permanent linkage and/or premature release of cytotoxic payloads. Herein, we developed a prodrug–antibody conjugate (ProADC) strategy by conjugating a bioorthogonal-activatable prodrug with an antibody that allowed on-target release and on-demand activation of cytotoxic drugs at a tumor site. The bioorthogonal-caged prodrug exhibited an enhanced permeability into and on-demand activation within cancer cells, while the pH-sensitive ADC linker allowed on-target release of the anticancer agent. Together, the ProADCs showed enhanced tumor penetration and alleviated side effects for use as an on-target and on-demand chemotherapy agents.

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.

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 Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin.

1990 ◽  
Vol 265 (24) ◽  
pp. 14450-14456 ◽  
Author(s):  
W.A. Palmisano ◽  
C.L. Treviño ◽  
M.T. Henzl
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Site Specific ◽  
Binding Loop

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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