- In situ preparation of protein - "Smart" polymer conjugates with retention of bioactivity
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Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by μLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by 1H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed.
- Heredia, Karina L.,Bontempo, Debora,Ly, Tiffany,Byers, Joshua T.,Halstenberg, Sven,Maynard, Heather D.
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- Synthesis, characterization, and in vivo delivery of siRNA-squalene nanoparticles targeting fusion oncogene in papillary thyroid carcinoma
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We report the conjugation of the natural lipid squalene (SQ) with a small interfering RNA (siRNA), against the junction oncogene RET/PTC1, usually found in papillary thyroid carcinoma (PTC). The acyclic isoprenoid chain of squalene has been covalently coupled with siRNA RET/PTC1 at the 3′-terminus of the sense strand via maleimide-sulfhydryl chemistry. Remarkably, the linkage of siRNA RET/PTC1 to squalene led to an amphiphilic molecule that self-organized in H2O as siRNA-SQ RET/PTC1 nanoparticles (NPs). The siRNA-SQ RET/PTC1 NPs, stable in H2O, were used for biological studies. In vitro, they did not show any cytotoxicity. Interestingly, in vivo, on a mice xenografted RET/PTC1 experimental model, RET/PTC1-SQ NPs were found to inhibit tumor growth and RET/PTC1 oncogene and oncoprotein expression after 2.5 mg/kg cumulative dose intravenous injections. In conclusion, these results showed that the "squalenoylation" offers a new noncationic plate-form for the siRNA delivery.
- Raouane, Mouna,Desmaele, Didier,Gilbert-Sirieix, Marie,Gueutin, Claire,Zouhiri, Fatima,Bourgaux, Claudie,Lepeltier, Elise,Gref, Ruxandra,Ben Salah, Ridha,Clayman, Gary,Massaad-Massade, Liliane,Couvreur, Patrick
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- Novel silyl ether-based acid-cleavable antibody-MMAE conjugates with appropriate stability and efficacy
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Antibody-drug conjugate (ADC) is a novel efficient drug delivery system that has been successfully used in clinical practice, and it has become a research hotspot in the anti-tumor drug field. Acid-cleavable linkers were first used in clinical ADCs, but their structural variety (e.g., hydrazone and carbonate) is still limited, and their stability is usually insufficient. Designing novel acid-cleavable linkers for the conjugation of the popular cytotoxin monomethyl auristatin E (MMAE) has always been a significant topic. In this paper, we generate a novel, silyl ether-based acid-cleavable antibody-MMAE conjugate, which skillfully achieves efficient combination of amino-conjugated MMAE with the acid-triggered silyl ether group by introducing p-hydroxybenzyl alcohol (PHB). The stability, acid-dependence cleavage, effective mechanism, efficacy and safety of the resulting ADC were systematically studied; the results show that it exhibits a significant improvement in stability, while maintaining appropriate efficacy and controlled therapeutic toxicity. This strategy is expected to expand a new type of acid-cleavable linkers for the development of ADCs with highly potent payloads.
- Wang, Yanming,Fan, Shiyong,Xiao, Dian,Xie, Fei,Li, Wei,Zhong, Wu,Zhou, Xinbo
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- Linker, linker-containing antibody conjugate drug and application of linker
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The invention relates to a linker as shown in a formula I, a linker-containing antibody conjugate drug and an application of the linker, also relates to a pharmaceutical composition comprising the antibody conjugate drug, and also relates to an application of the antibody conjugate drug to the treatment and/or prevention of diseases.
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Paragraph 0171-0173; 0183; 0187-0188
(2020/02/20)
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- RELEASABLE CONJUGATES
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The present application provides compounds of Formula (B), or pharmaceutically acceptable salts thereof, wherein D is a residue of a biologically active drug, which underdo hydrolysis under physiological conditions to release the biologically active drug and which are useful in the treatment of disorders that could be beneficially treated with the drug.
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Page/Page column 193; 194; 195
(2018/09/28)
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- Design, synthesis, and evaluation of linker-duocarmycin payloads: Toward selection of HER2-targeting antibody-drug conjugate SYD985
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Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.
- Elgersma, Ronald C.,Coumans, Ruud G. E.,Huijbregts, Tijl,Menge, Wiro M. P. B.,Joosten, John A. F.,Spijker, Henri J.,De Groot, Franciscus M. H.,Van Der Lee, Miranda M. C.,Ubink, Ruud,Van Den Dobbelsteen, Diels J.,Egging, David F.,Dokter, Wim H. A.,Verheijden, Gijs F. M.,Lemmens, Jacques M.,Timmers, C. Marco,Beusker, Patrick H.
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p. 1813 - 1835
(2015/06/16)
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- CELL PROLIFERATION INHIBITORS AND CONJUGATES THEREOF
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Disclosed herein are immunoconjugates comprising an inhibitor of Eg5 linked to an antigen binding moiety such as an antibody, useful for treating cell proliferative disorders. Also disclosed are novel inhibitors of Eg5 that can be used either alone or as part of an immunoconjugate to treat cell proliferation disorders. The Eg5 inhibitors include compounds of this formula as described herein: [insert last structure from page 68 here] The invention further provides pharmaceutical compositions comprising these compounds and immunoconjugates, optionally including a therapeutic co-agent, and methods to use these compounds, conjugates and compositions for treating cell proliferation disorders.
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Page/Page column 271
(2014/10/04)
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- Functionalized phosphonated half-cage molecules as ligands for metal complexes
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Phosphonated molecules, featuring a half-cage structure and a N-lateral chain with additional metal coordinating groups were designed as ligands of metal cations. These compounds were synthesized by a Diels-Alder (DA) strategy, using 1-diethoxyphosphoryl-1,3-butadiene and a series of N-substituted maleimides as dienophiles. Two cycloadducts, bearing a terminal primary alcohol and a terminal iodide, respectively, were used as key intermediates for further functionalizations. Metal coordination properties of the ligands equipped with functionalized N-lateral chains were proven by an ESI-HRMS study. The stoichiometry of one selected EuIII complex with a diphosphonated ligand was determined by photoluminescence spectroscopy in emission mode.
- Villemin, Elise,Herent, Marie-France,Marchand-Brynaert, Jacqueline
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supporting information
p. 6165 - 6178
(2013/01/15)
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- Temperature-regulated activity of responsive polymer-protein conjugates prepared by grafting-from via RAFT polymerization
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A facile route to well-defined "smart" polymer-protein conjugates with tunable bioactivity is reported. Protein modification with a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent room temperature polymerization in aqueous media led to conjugates of poly(N-isopropylacrylamide) and a model protein. Representing the first example of polymer-protein conjugation with RAFT agent immobilization via the "R-group" approach, high molecular weight and reductively stable conjugates were accessible without extensive purification or adverse effects on the protein structure. An increase in molecular weight with conversion was observed for the chains grafted from the protein surface, confirming the controlled nature of the polymerization. The responsive behavior of the immobilized polymer facilitated conjugate isolation and also allowed environmental modulation of bioactivity. Copyright
- De, Priyadarsi,Li, Ming,Gondi, Sudershan R.,Sumerlin, Brent S.
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supporting information; experimental part
p. 11288 - 11289
(2009/02/05)
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- The design, synthesis, and evaluation of two universal doxorubicin-linkers: Preparation of conjugates that retain topoisomerase II activity
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The design, synthesis, and evaluation of two N-alkylmaleimide aldehydes have been achieved, which upon reductive alkylation with the C3′-amino group of doxorubicin (DOX) permits the preparation of DOX conjugates via Michael addition of thiol-containing vectors. This method enables the mild, facile, and high-throughput preparation of DOX conjugates that retain the basic C3′-nitrogen, a pre-requisite for topoisomerase II inhibition. Seven DOX-amino acid conjugates were prepared, each displaying similar inhibitory activity as the parent drug.
- Sun, Chengzao,Aspland, Simon E.,Ballatore, Carlo,Castillo, Rosario,Smith III, Amos B.,Castellino, Angelo J.
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p. 104 - 107
(2007/10/03)
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