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.

Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format

2019 ◽  
Vol 30 (3) ◽  
pp. 698-702 ◽  
Author(s):  
Satoshi Kishimoto ◽  
Yuichi Nakashimada ◽  
Riri Yokota ◽  
Takaaki Hatanaka ◽  
Motoyasu Adachi ◽  
...  
Keyword(s):  
Therapeutic Antibody ◽  
Specific Chemical ◽  
Site Specific ◽  
Chemical Conjugation ◽  
Affinity Peptide

scholarly journals Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

2020 ◽  
Author(s):  
Camille M. Le Gall ◽  
Johan M.S. van der Schoot ◽  
Iván Ramos-Tomillero ◽  
Melek Parlak Khalily ◽  
Floris J. van Dalen ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Therapeutic Index ◽  
Stable Cell Line ◽  
Hybridoma Cells ◽  
Target Cells ◽  
Site Specific ◽  
Drug Conjugates ◽  
High Yields ◽  
A Site

I.AbstractFunctionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab’ fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab’ molecule (DTFab’), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab’ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

scholarly journals Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage φC31 Integrase

2001 ◽  
Vol 21 (12) ◽  
pp. 3926-3934 ◽  
Author(s):  
Bhaskar Thyagarajan ◽  
Eric C. Olivares ◽  
Roger P. Hollis ◽  
Daniel S. Ginsburg ◽  
Michele P. Calos
Keyword(s):  
Mammalian Cells ◽  
Chromosome Engineering ◽  
Site Specific ◽  
Site Specific Integration ◽  
Attachment Sites ◽  
Phage Integrase ◽  
Φc31 Integrase ◽  
Efficient Integration ◽  
A Site ◽  
Human And Mouse

ABSTRACT We previously established that the phage φC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phageattP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native “pseudo”attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

Clustered nanobody–drug conjugates for targeted cancer therapy

2020 ◽  
Vol 56 (65) ◽  
pp. 9344-9347
Author(s):  
Tiantian Wu ◽  
Manman Liu ◽  
Hai Huang ◽  
Yaping Sheng ◽  
Haihua Xiao ◽  
...  
Keyword(s):  
Side Effects ◽  
Cancer Therapy ◽  
Drug Efficacy ◽  
Targeted Cancer Therapy ◽  
Site Specific ◽  
Drug Conjugate ◽  
Drug Conjugates ◽  
A Site

A novel clustered nanobody–drug conjugate is constructed by a site-specific ligation of a nanobody to dendrimeric-lysine, which enhances drug efficacy and reduces side-effects.

Gram-Scale Antibody–Drug Conjugate Synthesis by Site-Specific Chemical Conjugation: AJICAP First Generation

2019 ◽  
Vol 23 (12) ◽  
pp. 2647-2654 ◽  
Author(s):  
Yutaka Matsuda ◽  
Kei Yamada ◽  
Tatsuya Okuzumi ◽  
Brian A. Mendelsohn
Keyword(s):  
First Generation ◽  
Antibody Drug Conjugate ◽  
Specific Chemical ◽  
Site Specific ◽  
Drug Conjugate ◽  
Chemical Conjugation ◽  
Antibody Drug

scholarly journals Good Manufacturing Practice Strategy for Antibody–Drug Conjugate Synthesis Using Site-Specific Chemical Conjugation: First-Generation AJICAP

ACS Omega ◽  
2019 ◽  
Vol 4 (24) ◽  
pp. 20564-20570 ◽  
Author(s):  
Yutaka Matsuda ◽  
Colin Clancy ◽  
Zhala Tawfiq ◽  
Veronica Robles ◽  
Brian A. Mendelsohn
Keyword(s):  
First Generation ◽  
Good Manufacturing Practice ◽  
Antibody Drug Conjugate ◽  
Specific Chemical ◽  
Site Specific ◽  
Drug Conjugate ◽  
Chemical Conjugation ◽  
Antibody Drug

Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

2011 ◽  
Vol 414 (2) ◽  
pp. 312-314 ◽  
Author(s):  
Erik Vernet ◽  
Jørgen Sauer ◽  
Andreas Andersen ◽  
Knud J. Jensen ◽  
Bjørn Voldborg
Keyword(s):  
Protein Expression ◽  
Specific Chemical ◽  
Site Specific ◽  
Ligation Independent Cloning ◽  
Chemical Conjugation

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.

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