scholarly journals Design and Validation of Linkers for Site-Specific Preparation of Antibody–Drug Conjugates Carrying Multiple Drug Copies Per Cysteine Conjugation Site

2020 ◽  
Vol 21 (18) ◽  
pp. 6882
Author(s):  
Amit Kumar ◽  
Shenlan Mao ◽  
Nazzareno Dimasi ◽  
Changshou Gao
Keyword(s):  
Alder Reaction ◽  
In Vitro Cytotoxicity ◽  
Multiple Drug ◽  
Drug Load ◽  
Site Specific ◽  
High Drug ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

First-generation cysteine-based site-specific antibody–drug conjugates (ADCs) are limited to one drug per cysteine. However, certain applications require a high drug to antibody ratio (DAR), such as when low-potency payloads are used. Higher drug load can be achieved using classical cysteine conjugation methods, but these result in heterogeneity, suboptimal efficacy and pharmacokinetics. Here, we describe the design, synthesis and validation of heterobifunctional linkers that can be used for the preparation of ADCs with a DAR of two, three and four in a site-specific manner per single cysteine conjugation site, resulting in site-specific ADCs with a DAR of four, six and eight. The designed linkers carry a sulfhydryl-specific iodoacetyl reactive group, and multiple cyclic diene moieties which can efficiently react with maleimide-carrying payloads through the Diels–Alder reaction. As a proof of concept, we synthesized site-specific DAR four, six and eight ADCs carrying tubulysin (AZ13601508) using engineered antibodies with a cysteine inserted after position 239 in the antibody CH2 domain. We evaluated and compared the in vitro cytotoxicity of ADCs obtained via the site-specific platform described herein, with ADCs prepared using classical cysteine conjugation. Our data validated a novel cysteine-based conjugation platform for the preparation of site-specific ADCs with high drug load for therapeutic applications.

scholarly journals Site-Specific Antibody–Drug Conjugates in Triple Variable Domain Fab Format

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 764 ◽  
Author(s):  
Dobeen Hwang ◽  
Christoph Rader
Keyword(s):  
Tumor Tissue ◽  
In Vitro Cytotoxicity ◽  
Variable Domain ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Solid Malignancies ◽  
Antibody Drug

The interest in replacing the conventional immunoglobulin G (IgG) format of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) with alternative antibody and antibody-like scaffolds reflects a need to expand their therapeutic utility and potency while retaining their exquisite specificity, affinity, and low intrinsic toxicity. For example, in the therapy of solid malignancies, the limited tumor tissue penetration and distribution of ADCs in IgG format mitigates a uniform distribution of the cytotoxic payload. Here, we report triple variable domain Fab (TVD–Fab) as a new format that affords the site-specific and stable generation of monovalent ADCs without the Fc domain and a drug-to-antibody ratio (DAR) of 2. TVD–Fabs harbor three variable fragment (Fv) domains: one for tumor targeting and two for the fast, efficient, precise, and stable conjugation of two cargos via uniquely reactive lysine residues. The biochemical and in vitro cytotoxicity properties of a HER2-targeting TVD–Fab before and after conjugation to a tubulin inhibitor were validated. In vivo, the TVD–Fab antibody carrier revealed a circulatory half-life of 13.3 ± 2.5 h and deeper tumor tissue distribution compared to our previously reported dual variable domain (DVD)–IgG1 format. Taken together, the TVD–Fab format merits further investigations as an antibody carrier of site-specific ADCs targeting solid malignancies.

Design, Synthesis, and in vitro Evaluation of Multivalent Drug Linkers for High-Drug-Load Antibody-Drug Conjugates

ChemMedChem ◽  
2018 ◽  
Vol 13 (8) ◽  
pp. 790-794 ◽  
Author(s):  
Bo Chen ◽  
Diego A. Gianolio ◽  
James E. Stefano ◽  
Charlene M. Manning ◽  
Richard C. Gregory ◽  
...  
Keyword(s):  
Drug Load ◽  
Design Synthesis ◽  
In Vitro Evaluation ◽  
High Drug ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

scholarly journals Synthesis of site-specific antibody-drug conjugates by ADP-ribosyl cyclases

2020 ◽  
Vol 6 (23) ◽  
pp. eaba6752 ◽  
Author(s):  
Zhefu Dai ◽  
Xiao-Nan Zhang ◽  
Fariborz Nasertorabi ◽  
Qinqin Cheng ◽  
Jiawei Li ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Reaction Times ◽  
Single Step ◽  
Her2 Positive ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Most of the current antibody-drug conjugates (ADCs) in clinic are heterogeneous mixtures. To produce homogeneous ADCs, established procedures often require multiple steps or long reaction times. The introduced mutations or foreign sequences may cause high immunogenicity. Here, we explore a new concept of transforming CD38 enzymatic activity into a facile approach for generating site-specific ADCs. This was achieved through coupling bifunctional antibody-CD38 fusion proteins with designer dinucleotide-based covalent inhibitors with stably attached payloads. The resulting adenosine diphosphate–ribosyl cyclase–enabled ADC (ARC-ADC) with a drug-to-antibody ratio of 2 could be rapidly generated through single-step conjugation. The generated ARC-ADC targeting human epidermal growth factor receptor 2 (HER2) displays excellent stability and potency against HER2-positive breast cancer both in vitro and in vivo. This proof-of-concept study demonstrates a new strategy for production of site-specific ADCs. It may provide a general approach for the development of a novel class of ADCs with potentially enhanced properties.

scholarly journals In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

PLoS ONE ◽  
2014 ◽  
Vol 9 (1) ◽  
pp. e83865 ◽  
Author(s):  
Dowdy Jackson ◽  
John Atkinson ◽  
Claudia I. Guevara ◽  
Chunying Zhang ◽  
Vladimir Kery ◽  
...  
Keyword(s):  
In Vivo Evaluation ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading

2015 ◽  
Vol 33 (7) ◽  
pp. 694-696 ◽  
Author(s):  
Pavel Strop ◽  
Kathy Delaria ◽  
Davide Foletti ◽  
Jody Melton Witt ◽  
Adela Hasa-Moreno ◽  
...  
Keyword(s):  
Drug Loading ◽  
Therapeutic Index ◽  
Site Specific ◽  
High Drug ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
High Drug Loading ◽  
Antibody Drug

Impact of cathepsin B-sensitive triggers and hydrophilic linkers onin vitroefficacy of novel site-specific antibody–drug conjugates

2018 ◽  
Vol 16 (11) ◽  
pp. 1882-1889 ◽  
Author(s):  
Francesca Bryden ◽  
Camille Martin ◽  
Stéphanie Letast ◽  
Eva Lles ◽  
Inmaculada Viéitez-Villemin ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Cathepsin B ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Promising anti-HER2 site-specific ADCs with anin vitroefficacy equivalent to Kadcyla®.

scholarly journals Characterization and in vitro data of antibody drug conjugates (ADCs) derived from heterotrifunctional linker designed for the site-specific preparation of dual ADCs

Data in Brief ◽  
2018 ◽  
Vol 21 ◽  
pp. 2208-2220 ◽  
Author(s):  
Amit Kumar ◽  
Krista Kinneer ◽  
Luke Masterson ◽  
Ebele Ezeadi ◽  
Philip Howard ◽  
...  
Keyword(s):  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Vitro Data ◽  
In Vitro Data ◽  
Antibody Drug

Abstract B130: Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells

2015 ◽  
Author(s):  
Rajeeva Singh ◽  
Paulin Salomon ◽  
Yulius Setiady ◽  
Jose Ponte
Keyword(s):  
Cancer Cells ◽  
In Vitro Cytotoxicity ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug ◽  
Target Cancer

A site-specific approach to improved antibody-drug conjugates.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13009-e13009 ◽  
Author(s):  
James Italia
Keyword(s):  
Target Identification ◽  
Scale Up ◽  
Full Potential ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Safety Studies ◽  
A Site ◽  
Antibody Drug

e13009 Background: Antibody based therapeutics have emerged as a highly promising treatment option for various forms of cancer. One such class of emergent antibody therapeutics is antibody-drug conjugates (ADCs), which has experienced remarkable success in the past few decades, including many candidates currently undergoing clinical trials. Despite its promise, developing effective ADCs remains a challenge due to extensive optimization with regard to antibody target identification, conjugate specificity, conjugation chemistry compatibility, heterogeneous side product co-purification, all affecting efficacy, PK/PD characteristics, and toxicity. There is a pressing need to develop alternative strategies for precise (site-specific), flexible (offering numerous options for conjugation site and chemistry), efficient, and scalable generation of antibody-drug conjugates to overcome these limitations and realize the full potential of this highly promising class of therapeutics. Methods: In this study, we demonstrate the feasibility of efficiently incorporating an azide-containing UAA into full-length antibody at different site and facilitate its site-specific functionalization, which provides a facile scale-up compatible route to generate precise antibody-drug conjugates with fine-tuned therapeutic properties. Specifically, we have engineered multiple variants of trastuzumab to contain the aforementioned UAA, enabling site-specific attachment of cytotoxic payloads, such as MMAE/MMAF, via click chemistry to generate ADCs with homogeneous DARs. Results: The resulting BrickBio ADCs exhibit high therapeutic efficacy against HER2+ cell lines in vitro as well as in mouse xenograft models. These homogenous ADCs additionally outperform traditional antibody conjugation technologies which lack site-specificity or site-selectivity. Conclusions: The ability to systematically vary the attachment topology between the drug and the antibody is a major strength of this approach and preliminary data suggest that such variation indeed affect ADC performance. BrickBio ADCs are currently in the preclinical stage with further efforts for safety studies underway.

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