scholarly journals Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues

2021 ◽  
Vol 14 (7) ◽  
pp. 672
Author(s):  
Qun Zhou ◽  
Josephine Kyazike ◽  
Ekaterina Boudanova ◽  
Michael Drzyzga ◽  
Denise Honey ◽  
...  
Keyword(s):  
Specific Antibody ◽  
High Selectivity ◽  
Therapeutic Index ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Site Specific ◽  
Drug Conjugates ◽  
High Therapeutic Index ◽  
High Site ◽  
Antibody Conjugation

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

scholarly journals Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

2019 ◽  
Author(s):  
Susanna K. Elledge ◽  
Hai L. Tran ◽  
Alec H. Christian ◽  
Veronica Steri ◽  
Byron Hann ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Xenograft Model ◽  
Antibody Engineering ◽  
Antibody Drug Conjugate ◽  
Site Specific ◽  
Drug Conjugates ◽  
Mouse Xenograft Model ◽  
Random Modification ◽  
The Stability ◽  
Antibody Drug

AbstractChemical modification of antibodies is one of the most important bioconjugations utilized by biologists and biotechnology. To date, the field has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently we have developed oxaziridine chemistry for highly selective and efficient sulfimide modification of methionine called redox-activated chemical tagging (ReACT). Here, we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and generally good reaction efficiencies with the panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than ten-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, likely because of their reduced access to water. There was also a ten-fold variation in stability depending on the nature of the oxaziridine, which we determined was inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs and antibody drug conjugate potencies were comparable to those reported for cysteine-maleimide modifications of trastuzumab. We also found our antibody drug conjugates to be potent in a breast cancer mouse xenograft model. These studies provide a roadmap for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation.

Abstract 752: CovisoLinK: New bacterial transglutaminase Q-Tag substrate for the development of site specific antibody-drug conjugates

2018 ◽  
Author(s):  
Eva Sivado ◽  
Vincent Thomas ◽  
Meddy El Alaoui ◽  
Anne-Catherine Jallas ◽  
Mike R. Dyson ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Proximity-Induced Site-Specific Antibody Conjugation

2018 ◽  
Vol 29 (11) ◽  
pp. 3522-3526 ◽  
Author(s):  
Chenfei Yu ◽  
Juan Tang ◽  
Axel Loredo ◽  
Yuda Chen ◽  
Sung Yun Jung ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Site Specific ◽  
Antibody Conjugation

Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody–Drug Conjugates

2016 ◽  
Vol 138 (4) ◽  
pp. 1430-1445 ◽  
Author(s):  
Jeffrey C. Kern ◽  
Mark Cancilla ◽  
Deborah Dooney ◽  
Kristen Kwasnjuk ◽  
Rena Zhang ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

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 antibody drug conjugates for cancer therapy

mAbs ◽  
2013 ◽  
Vol 6 (1) ◽  
pp. 34-45 ◽  
Author(s):  
Siler Panowski ◽  
Sunil Bhakta ◽  
Helga Raab ◽  
Paul Polakis ◽  
Jagath R Junutula
Keyword(s):  
Cancer Therapy ◽  
Specific Antibody ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Site-specific antibody-drug conjugates with variable drug-to-antibody-ratios for AML therapy

2021 ◽  
Author(s):  
Zhefu Dai ◽  
Xiao-Nan Zhang ◽  
Qinqin Cheng ◽  
Fan Fei ◽  
Tianling Hou ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug
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