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 Thermo-Sensitive Vesicles in Controlled Drug Delivery for Chemotherapy

Pharmaceutics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 150 ◽  
Author(s):  
Elisabetta Mazzotta ◽  
Lorena Tavano ◽  
Rita Muzzalupo
Keyword(s):  
Drug Delivery ◽  
Cancer Treatment ◽  
Controlled Drug Delivery ◽  
Solid Tumours ◽  
Site Specific ◽  
Mild Hyperthermia ◽  
Promising Tool ◽  
A Site

Thermo-sensitive vesicles are a promising tool for triggering the release of drugs to solid tumours when used in combination with mild hyperthermia. Responsivity to temperature makes them intelligent nanodevices able to provide a site-specific chemotherapy. Following a brief introduction concerning hyperthermia and its advantageous combination with vesicular systems, recent investigations on thermo-sensitive vesicles useful for controlled drug delivery in cancer treatment are reported in this review. In particular, the influence of bilayer composition on the in vitro and in vivo behaviour of thermo-sensitive formulations currently under investigation have been extensively explored.

scholarly journals TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

2007 ◽  
Vol 189 (24) ◽  
pp. 9037-9043 ◽  
Author(s):  
Carolina Elvira César ◽  
Matxalen Llosa
Keyword(s):  
Promoter Sequence ◽  
Dna Topology ◽  
Integration Host Factor ◽  
Host Factors ◽  
Elongation Complex ◽  
Site Specific ◽  
Double Stranded Dna ◽  
Transcript Elongation ◽  
A Site

ABSTRACT R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a “closed” conformation. In an IHF-deficient (IHF−) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF− background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an “open” form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

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 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 Gene therapy: advances, challenges and perspectives

2017 ◽  
Vol 15 (3) ◽  
pp. 369-375 ◽  
Author(s):  
Giulliana Augusta Rangel Gonçalves ◽  
Raquel de Melo Alves Paiva
Keyword(s):  
Gene Therapy ◽  
Germ Line ◽  
Genetic Material ◽  
The United States ◽  
In Vivo Studies ◽  
Basic Unit ◽  
Target Cells ◽  
Site Specific ◽  
Genomic Editing

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

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.

scholarly journals Investigations of human myosin VI targeting using optogenetically controlled cargo loading

2017 ◽  
Vol 114 (9) ◽  
pp. E1607-E1616 ◽  
Author(s):  
Alexander R. French ◽  
Tobin R. Sosnick ◽  
Ronald S. Rock
Keyword(s):  
Avena Sativa ◽  
Activation Mechanism ◽  
Myosin Vi ◽  
Site Specific ◽  
Specific Location ◽  
Cargo Protein ◽  
Specific Manner ◽  
A Site

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.

scholarly journals Flp1 may function in the resolution of recombinant DNA intermediates

2019 ◽  
Vol 15 (3) ◽  
pp. 109
Author(s):  
Phung Thi Thu Huong ◽  
Tran Hong Diem ◽  
Nguyen Luong Hieu Hoa ◽  
Vo Thanh Sang ◽  
Le Van Minh ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Drug Sensitivity ◽  
Recombinant Dna ◽  
Alternative Pathway ◽  
Single Copy ◽  
Site Specific ◽  
Dna Damaging Agents ◽  
2 Micron Plasmid ◽  
A Site

Mus81 is a structure-selective endonuclease which constitutes an alternative pathway in parallel with the helicase-topoisomerase Sgs1-Top3-Rmi1 complex to resolve a number of DNA intermediates during DNA replication, repair, and homologous recombination. Previously, it was shown that the N-terminal region of Mus81 was required for its in vivo function in a redundant manner with Sgs1; sgs1Δmus81Δ100N cells are sensitive to DNA damaging agents. In this study, a single-copy suppressor screening to seek for a factor(s) that could rescue the drug sensitivity of sgs1Δmus81Δ100N cells was performed and revealed that Flp1, a site-specific recombinase 1 encoded on the 2-micron plasmid was a suppressor. This result suggests a function of Flp1 in coordination with Mus81 and Sgs1 to resolve the recombinant DNA intermediates.

scholarly journals Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody–Drug Conjugates

2021 ◽  
Author(s):  
Yutaka Matsuda ◽  
Takuya Seki ◽  
Kei Yamada ◽  
Yuri Ooba ◽  
Kazutoshi Takahashi ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

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
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