RESUMO
Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.
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GlycoConnect technology can be readily adapted to provide different drug-to-antibody ratios (DARs) and is currently also evaluated in various clinical programs, including ADCT-601 (DAR2), MRG004a (DAR4), and XMT-1660 (DAR6). While antibody-drug conjugates (ADCs) typically feature a DAR2-8, it has become clear that ADCs with ultrapotent payloads (e.g., PBD dimers and calicheamicin) can only be administered to patients at low doses (<0.5 mg/kg), which may compromise effective biodistribution and may be insufficient to reach target receptor saturation in the tumor. Here, we show that GlycoConnect technology can be readily extended to DAR1 ADCs without the need of antibody re-engineering. We demonstrate that various ultrapotent, cytotoxic payloads are amenable to this methodology. In a follow-up experiment, HCC-1954 tumor spheroids were treated with either an AlexaFluor647-labeled DAR1 or DAR2 PBD-based ADC to study the effect on tumor penetration. Significant improvement of tumor spheroid penetration was observed for the DAR1 ADC compared to the DAR2 ADC at an equal payload dose, underlining the potential of a lower DAR for ADCs bearing ultrapotent payloads.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Imunoconjugados , Neoplasias Hepáticas , Humanos , Imunoconjugados/uso terapêutico , Distribuição Tecidual , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , TecnologiaRESUMO
Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Linfócitos T , Química Click , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/química , Antígenos de Neoplasias/metabolismoRESUMO
Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinone were quantified by stopped flow UV-Vis spectroscopy and computational analysis. We found that the strained alkyne BCN-OH 3 (k2 1824â M-1 s-1 ) reacts >150â times faster than the strained alkene TCO-OH 5 (k2 11.56â M-1 s-1 ), and that derivatization with a carbamate can lead to a reduction of the rate constant with almost half. Also, the 8-membered strained alkyne BCN-OH 3 reacts 16â times faster than the more strained 7-membered THS 2 (k2 110.6â M-1 s-1 ). Using the linearized Eyring equation we determined the thermodynamic activation parameters of these two strained alkynes, revealing that the SPOCQ reaction of quinone 1 with THS 2 is associated with ΔH≠ of 0.80â kcal/mol, ΔS≠ =-46.8 cal/Kâ mol, and ΔG≠ =14.8â kcal/mol (at 25 °C), whereas the same reaction with BCN-OH 3 is associated with, ΔH≠ =2.25â kcal/mol, ΔS≠ =-36.3 cal/Kâ mol, and ΔG≠ =13.1â kcal/mol (at 25 °C). Computational analysis supported the values obtained by the stopped-flow measurements, with calculated ΔG≠ of 15.6â kcal/mol (in H2 O) for the SPOCQ reaction with THS 2, and with ΔG≠ of 14.7â kcal/mol (in H2 O) for the SPOCQ reaction with BCN-OH 3. With these empirically determined thermodynamic parameters, we set an important step towards a more fundamental understanding of this set of rapid click reactions.
RESUMO
Click chemistry has been established rapidly as one of the most valuable methods for the chemical transformation of complex molecules. Due to the rapid rates, clean conversions to the products, and compatibility of the reagents and reaction conditions even in complex settings, it has found applications in many molecule-oriented disciplines. From the vast landscape of click reactions, approaches have emerged in the past decade centered around oxidative processes to generate in situ highly reactive synthons from dormant functionalities. These approaches have led to some of the fastest click reactions know to date. Here, we review the various methods that can be used for such oxidation-induced "one-pot" click chemistry for the transformation of small molecules, materials, and biomolecules. A comprehensive overview is provided of oxidation conditions that induce a click reaction, and oxidation conditions are orthogonal to other click reactions so that sequential "click-oxidation-click" derivatization of molecules can be performed in one pot. Our review of the relevant literature shows that this strategy is emerging as a powerful approach for the preparation of high-performance materials and the generation of complex biomolecules. As such, we expect that oxidation-induced "one-pot" click chemistry will widen in scope substantially in the forthcoming years.
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The availability of tools to generate homogeneous and stable antibody conjugates without recombinant DNA technology is a valuable asset in fields spanning from in vitro diagnostics to in vivo imaging and therapeutics. We present here a general approach for the conjugation to human IgG1 antibodies, by employing a straightforward two-stage protocol based on antibody deglycosylation followed by tyrosinase-mediated ortho-quinone strain-promoted click chemistry. The technology is validated by the efficient and clean generation of highly potent DAR2 and DAR4 antibody-drug conjugates (ADCs) with cytotoxic payloads MMAE or PBD dimer, and their in vitro evaluation.
Assuntos
Trastuzumab , Tirosina , Anticorpos MonoclonaisRESUMO
Control over the placement and activity of biomolecules on solid surfaces is a key challenge in bionanotechnology. While covalent approaches excel in performance, physical attachment approaches excel in ease of processing, which is equally important in many applications. We show how the precision of recombinant protein engineering can be harnessed to design and produce protein-based diblock polymers with a silica-binding and highly hydrophilic elastin-like domain that self-assembles on silica surfaces and nanoparticles to form stable polypeptide brushes that can be used as a scaffold for later biofunctionalization. From atomic force microscopy-based single-molecule force spectroscopy, we find that individual silica-binding peptides have high unbinding rates. Nevertheless, from quartz crystal microbalance measurements, we find that the self-assembled polypeptide brushes cannot easily be rinsed off. From atomic force microscopy imaging and bulk dynamic light scattering, we find that the binding to silica induces fibrillar self-assembly of the peptides. Hence, we conclude that the unexpected stability of these self-assembled polypeptide brushes is at least in part due to peptide-peptide interactions of the silica-binding blocks at the silica surface.
Assuntos
Elastina , Nanopartículas , Elastina/genética , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Peptídeos , Dióxido de SilícioRESUMO
Fast, selective and facile functionalization of biologically relevant molecules is a pursuit of ever-growing importance. A promising approach in this regard employs the high reactivity of quinone and quinone analogues for fast conjugation chemistry by nucleophilic addition or cycloadditions. Combined with in situ generation of these compounds, selective conjugation on proteins and surfaces can be uniquely induced in a time and spatially resolved manner: generation of a quinone can often be achieved by simple addition of an enzyme or stoichiometric amounts of chemoselective oxidant, or by exposure to light. In this Minireview, we discuss the generation and subsequent functionalization of quinones, iminoquinones, and quinone methides. We also discuss practical applications regarding these conjugation strategies.
RESUMO
Stimulated by its success in both bioconjugation and surface modification, we studied the strain-promoted oxidation-controlled cycloalkyne-1,2-quinone cycloaddition (SPOCQ) in three ways. First, the second-order rate constants and activation parameters (ΔH⧧) were determined of various cyclooctynes reacting with 4-tert-butyl-1,2-quinone in a SPOCQ reaction, yielding values for ΔH⧧ of 4.5, 7.3, and 12.1 kcal/mol, for bicyclo[6.1.0]non-4-yne (BCN), cyclooctyne (OCT), and dibenzoazacyclooctyne (DIBAC), respectively. Second, their reaction paths were investigated in detail by a range of quantum mechanical calculations. Single-configuration theoretical methods, like various DFT and a range of MP2-based methods, typically overestimate this barrier by 3-8 kcal/mol (after inclusion of zero-point energy, thermal, and solvation corrections), whereas MP2 itself underestimates the barrier significantly. Only dispersion-corrected DFT methods like B97D (yielding 4.9, 6.4, and 12.1 kcal/mol for these three reactions) and high-level CCSD(T) and multireference multiconfiguration AQCC ab initio approaches (both yielding 8.2 kcal/mol for BCN) give good approximations of experimental data. Finally, the multireference methods show that the radical character in the TS is rather small, thus rationalizing the use of single-reference methods like B97D and SCS-MP2 as intrinsically valid approaches.
RESUMO
Target-specific killing of tumor cells with antibody-drug conjugates (ADCs) is an elegant concept in the continued fight against cancer. However, despite more than 20 years of clinical development, only four ADC have reached market approval, while at least 50 clinical programs were terminated early. The high attrition rate of ADCs may, at least in part, be attributed to heterogeneity and instability of conventional technologies. At present, various (chemo)enzymatic approaches for site-specific and stable conjugation of toxic payloads are making their way to the clinic, thereby potentially providing ADCs with increased therapeutic window.
Assuntos
Enzimas/química , Imunoconjugados/química , Humanos , Relação Estrutura-AtividadeRESUMO
Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidation of tyrosine with mushroom tyrosinase, strain-promoted cycloaddition (SPOCQ) of the resulting 1,2-quinone with various bicyclo[6.1.0]nonyne (BCN) derivatives led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific preparation of an antibody-drug conjugate.
Assuntos
Imunoconjugados/química , Monofenol Mono-Oxigenase/química , Coloração e Rotulagem/métodos , Tirosina/química , Celulases , Reação de Cicloadição , OxirreduçãoRESUMO
Strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) between functionalized bicyclo[6.1.0]non-4-yne (BCN) and surface-bound quinones revealed an unprecedented 100 % conjugation efficiency. In addition, monitoring by direct analysis in real time mass spectrometry (DART-MS) revealed the underlying kinetics and activation parameters of this immobilization process in dependence on its microenvironment.
RESUMO
Mica is the substrate of choice for microscopic visualization of a wide variety of intricate nanostructures. Unfortunately, the lack of a facile strategy for its modification has prevented the on-mica assembly of nanostructures. Herein, we disclose a convenient catechol-based linker that enables various surface-bound metal-free click reactions, and an easy modification of mica with DNA nanostructures and a horseradish peroxidase mimicking hemin/G-quadruplex DNAzyme.
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Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by â¼50-60%, heparan sulfate by â¼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by â¼74 and â¼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by â¼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.
Assuntos
Acetilgalactosamina/análogos & derivados , Glicosaminoglicanos/biossíntese , Acetilgalactosamina/química , Acetilgalactosamina/farmacologia , Animais , Linhagem Celular , Embrião de Galinha , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos/antagonistas & inibidores , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Polissacarídeos/antagonistas & inibidores , Polissacarídeos/biossíntese , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a 'non-cleavable' bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free 'click' reaction, leading to a VPg-triazole-RNA construct that was 'non-cleavable' by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.
Assuntos
Peptídeos/química , Picornaviridae/genética , Biossíntese de Proteínas , RNA Viral/biossíntese , RNA Viral/química , Replicação Viral , Química Click , Enterovirus/genética , Genoma Viral , Células HeLa , Humanos , Picornaviridae/fisiologia , RNA/química , Proteínas Virais/químicaRESUMO
A main challenge in the area of bioconjugation is to devise reactions that are both activatable and fast. Here, we introduce a temporally controlled reaction between cyclooctynes and 1,2-quinones, induced by facile oxidation of 1,2-catechols. This so-called strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) shows a remarkably high reaction rate when performed with bicyclononyne (BCN), outcompeting the well-known cycloaddition of azides and BCN by 3 orders of magnitude, thereby allowing a new level of orthogonality in protein conjugation.
Assuntos
Alcinos/química , Catecóis/química , Proteínas/química , Quinonas/química , Azidas/química , Química Click , Ciclização , Reação de Cicloadição , Modelos MolecularesRESUMO
A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Polissacarídeos/química , Ado-Trastuzumab Emtansina , Anticorpos Monoclonais Humanizados/química , Azidas/química , Sequência de Carboidratos , Química Click , Glicosilação , Humanos , Maitansina/análogos & derivados , Maitansina/química , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Trastuzumab/químicaRESUMO
We have synthesized biologically relevant 6-aza-8-oxa[3.2.1]bicyclooctane scaffolds in a five-step procedure starting from furfural. Besides showing that these scaffolds are amenable to decoration via standard functional group interconversions, we also describe investigations for further functionalization via Lewis acid-mediated N,O-acetal opening, followed by nucleophilic trapping of the resulting intermediate cation. By using different nucleophiles, we have successfully prepared a modest library of 2,6-trans-disubstituted pyrans in good yields and in a highly diastereoselective manner.
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Acetais/química , Compostos Aza/química , Compostos Azo/química , Compostos Bicíclicos com Pontes/química , Descoberta de Drogas , Furaldeído/química , Catálise , Estrutura Molecular , EstereoisomerismoRESUMO
Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.
Assuntos
Western Blotting , Citrulina/química , Fenilglioxal/química , Proteínas/química , Animais , Western Blotting/métodos , Catálise , Humanos , Hidrolases/metabolismo , Indicadores e Reagentes/química , Desiminases de Arginina em Proteínas , Proteínas/metabolismo , Coloração e RotulagemRESUMO
The transient receptor potential vanilloid type 5 (TRPV5) Ca(2+) channel facilitates transcellular Ca(2+) transport in the distal convoluted tubule (DCT) of the kidney. The channel is glycosylated with a complex type N-glycan and it has been postulated that hydrolysis of the terminal sialic acid(s) stimulate TRPV5 activity. The present study delineates the role of the N-glycan in TRPV5 activity using biochemical assays in Human Embryonic Kidney 293 cells expressing TRPV5, isoelectric focusing and total internal reflection fluorescent microscopy. The anti-aging hormone klotho and other glycosidases stimulate TRPV5-dependent Ca(2+) uptake. Klotho was found to increase the plasma membrane stability of TRPV5, via the TRPV5 N-glycan. Sialidase mimicked this stimulatory action. However, this effect was independent of the N-glycosylation state of TRPV5, since the N-glycosylation mutant (TRPV5(N358Q)) was activated to the same extent. We showed that the increased TRPV5 activity after sialidase treatment is caused by inhibition of lipid raft-mediated internalization. In addition, sialidase modified the N-glycan of transferrin, a model glycoprotein, differently from klotho. Previous studies showed that after klotho treatment, galectin-1 binds the TRPV5 N-glycan and thereby increases TRPV5 activity. However, galectin-3, but not galectin-1, was expressed in the DCT. Furthermore, an increase in TRPV5-mediated Ca(2+) uptake was detected after galectin-3 treatment. In conclusion, two distinct TRPV5 stimulatory mechanisms were demonstrated; a klotho-mediated effect that is dependent on the N-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5.