RESUMEN
Conjugate vaccines prepared with the cross-reactive material 197 (CRM197) carrier protein have been successful in the clinic and are of great interest in the field of immunotherapy. One route to preparing peptide-CRM197 conjugate vaccines involves an activation-conjugation strategy, effectively coupling lysine residues on the protein to cysteine thiolate groups on the peptide of interest using a heterobifunctional linker as an activation agent. This method has been found to result in two distinct populations of conjugates, believed to be the result of a conformational change of CRM197 during preparation. This report explores the factors that lead to this conformational change, pointing to a model in which the unintentional alkylation of histidine-21 by the activating agent promotes the "opening" of the monomeric protein. This exposes a new set of lysine residues that are modified by additional activation agents. Subsequent peptide ligation to these sites results in the two conformers. This is the first time that a specific chemical modification is demonstrated to induce a defined conformational change for this carrier protein. Importantly, alternative conditions and reagents have been found to minimize this effect, improving the conformational homogeneity of peptide-CRM197 conjugates.
Asunto(s)
Proteínas Bacterianas/química , Péptidos/química , Vacunas Conjugadas/química , Conformación ProteicaRESUMEN
A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein-antibody conjugate, CVX-343, an extended half-life therapeutic for the treatment of metabolic disease. CVX-343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half-life extension. CVX-343 is representative of a growing number of complex novel peptide- and protein-based bioconjugate molecules currently being explored as therapeutic candidates. The complexity of these bioconjugates, assembled using well-established chemistries, can lead to very difficult production schemes requiring multiple starting materials and a combination of diverse technologies. Key improvements had to be made to the original CVX-343 Phase 1 manufacturing process in preparation for Phase 3 and commercial manufacturing. A strategy of minimizing FGF21A129C dimerization and stabilizing the FGF21A129C Drug Substance Intermediate (DSI), linker, and activated FGF21 intermediate was pursued. The use of tris(2-carboxyethyl)phosphine (TCEP) to prevent FGF21A129C dimerization through disulfide formation was eliminated. FGF21A129C dimerization and linker hydrolysis were minimized by formulating and activating FGF21A129C at acidic instead of neutral pH. An activation use test was utilized to guide FGF21A129C pooling in order to minimize misfolds, dimers, and misfolded dimers in the FGF21A129C DSI. After final optimization of reaction conditions, a process was established that reduced the consumption of FGF21A129C by 36% (from 4.7 to 3.0 equivalents) and the consumption of linker by 55% (from 1.4 to 0.95 equivalents for a smaller required amount of FGF21A129C ). The overall process time was reduced from â¼5 to â¼3 days. The product distribution improved from containing â¼60% to â¼75% desired bifunctionalized (+2 FGF21) FGF21-antibody conjugate in the crude conjugation mixture and from â¼80% to â¼85% in the final CVX-343 Drug Substance (DS), while maintaining the same overall process yield based on antibody scaffold input.
RESUMEN
Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.
Asunto(s)
Péptidos/síntesis química , Anticuerpos/química , Química Clic , Estructura Molecular , Péptidos/química , Péptidos/aislamiento & purificaciónRESUMEN
Angiogenesis is a multi-step process regulated by pro- and anti-angiogenic factors. Inhibition of angiogenesis is a potential anti cancer treatment strategy that is now investigated clinically. In addition, advances in the understanding of the angiogenic process have led to the development of new angiogenesis therapies for ischemic heart disease. Currently, researchers search for objective measures that indicate pharmacological responses to pro- and anti-angiogenic drugs and therefore, there is a great interest in techniques to visualize angiogenesis noninvasively. As CD13 is selectively expressed in angiogenic blood vessels, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in animal models and patients. Here, an overview on the currently used CD13 targeted molecular imaging probes for noninvasive visualization of angiogenesis is given.
Asunto(s)
Imagen Molecular , Sondas Moleculares/farmacología , Neovascularización Patológica/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Animales , Humanos , Modelos Moleculares , Conformación Molecular , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/químicaRESUMEN
Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.
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Antineoplásicos , Neoplasias de la Mama , Inmunoconjugados , Humanos , Femenino , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Anticuerpos , Línea Celular Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell-surface sialylated glycoproteins while maintaining high cell viability.
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Biotinilación/métodos , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Coloración y Etiquetado/métodos , Aldehídos/química , Compuestos de Anilina/química , Animales , Biotina/análogos & derivados , Biotina/química , Células CHO , Catálisis , Línea Celular , Supervivencia Celular , Cricetinae , Cricetulus , Endocitosis , Citometría de Flujo , Humanos , Antígenos Comunes de Leucocito/química , Microscopía Fluorescente , Ácido N-Acetilneuramínico/análogos & derivados , Oxidación-Reducción , Oximas/química , Ácido Peryódico/química , Sialiltransferasas/química , Estreptavidina/química , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.
Asunto(s)
Antígenos Bacterianos/química , Toxinas Bacterianas/química , Proteínas de Unión a Maltosa/química , Nanoestructuras/química , Nanotecnología/métodos , Proteínas Recombinantes/química , Virión/metabolismo , Compuestos de Anilina/química , Animales , Baculoviridae/química , Baculoviridae/genética , Baculoviridae/metabolismo , Western Blotting , Clonación Molecular , Escherichia coli , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Nanoestructuras/virología , Oximas/química , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Estereoisomerismo , Resonancia por Plasmón de Superficie , Transfección , Virión/genéticaRESUMEN
A high-yielding and rapid chemoselective ligation approach is presented that uses aniline catalysis to activate aromatic aldehydes toward two amine nucleophiles, namely, 6-hydrazinopyridyl and aminooxyacetyl groups. The rates of these ligations are resolved for model reactions with unprotected peptides. The resulting hydrazone and oxime conjugates are attained under ambient conditions with rate constants of 10(1)-10(3) M(-1) s(-1). These rate constants exceed those of current chemoselective ligation chemistries and enable efficient labeling of peptides and proteins at low muM concentrations, at neutral pH, without using a large excess of one of the components. The utility of the approach is demonstrated by the p-fluorobenzylation of human serum albumin and by the fluorescent labeling of an unprotected peptide with Alexa Fluor 488.
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Aldehídos/química , Hidrazonas/química , Oximas/química , Coloración y Etiquetado/métodos , Ácido Aminooxiacético/química , Compuestos de Anilina/química , Catálisis , Humanos , Oxidación-Reducción , Péptidos/metabolismo , Piridonas/química , Piridonas/metabolismo , Albúmina Sérica/metabolismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
A strategy for the synthesis of multivalent peptide-based nonsymmetric dendrimers by native chemical ligation using poly(lysine) dendritic wedges as scaffolds is presented.
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Dendrímeros/síntesis química , Péptidos/química , Polilisina/químicaRESUMEN
The synthesis of a cyclic peptide-Gd(III)DTPA molecule equipped with biotin is presented, yielding a well-defined multivalent MRI contrast agent after its coupling to avidin.
Asunto(s)
Medios de Contraste/química , Imagen por Resonancia Magnética/métodos , Neovascularización Fisiológica , Avidina/química , Biotina/química , Modelos Moleculares , Estructura Molecular , VolumetríaAsunto(s)
Hidrazonas/química , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Biotina/química , Biotina/metabolismo , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/aislamiento & purificación , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificación , Estreptavidina/química , Estreptavidina/metabolismoRESUMEN
[structure: see text] A bimodal target-specific contrast agent based on a cyclic peptide containing the target-specific NGR sequence, gadolinium(III) diethylenetriaminepentaacetic acid (Gd(III)DTPA), and Oregon Green 488 (OG488) suitable for both MR imaging and optical imaging of angiogenesis is developed. The synthetic strategy for this target-specific contrast agent exploits the use of highly efficient, chemoselective reactions, such as native chemical ligation, and gives a straightforward approach for double labeling of peptides in general.
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Ácidos Carboxílicos/química , Medios de Contraste/síntesis química , Gadolinio DTPA/química , Imagen por Resonancia Magnética/métodos , Neovascularización Patológica/diagnóstico , Péptidos Cíclicos/química , Medios de Contraste/química , Gadolinio DTPA/síntesis química , Estructura Molecular , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)2 (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)2 versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)2 with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.
RESUMEN
Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.
Asunto(s)
Comovirus/química , Nanopartículas/química , Péptidos de Penetración Celular/química , Portadores de Fármacos/química , Células HeLa , Humanos , Hidrazonas/química , Microscopía Confocal , Péptidos/química , Temperatura , TransfecciónRESUMEN
In this article, we introduce the use of a thiaproline-modified lysine side-chain [Lys(Thz)], as an unlockable handle that enables late-stage, site-selective modification of chemically synthesized proteins. The Lys(Thz) residue was incorporated into the murine chemokine RANTES to demonstrate its compatibility with Boc/Bzl solid phase peptide synthesis, native chemical ligation, and disulfide bond formation. After oxidative folding of the protein, the thiol was liberated under mild reaction conditions [0.2 M hydroxylamine (NH2OH) or O-methylhydroxylamine (MeONH2), pH 4] and was subsequently reacted with thiol-selective tags. This side chain protection strategy enables the use of readily available thiol-reactive probes for the modification of internally disulfide bonded proteins.
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Quimiocina CCL5/química , Disulfuros/química , Lisina/análogos & derivados , Lisina/química , Pliegue de Proteína , Animales , Ratones , Oxidación-ReducciónRESUMEN
OBJECTIVES: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal alpha(2)-antiplasmin-based contrast agent (CA). BACKGROUND: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links alpha(2)-antiplasmin to fibrin, indicating the potential of alpha(2)-antiplasmin-based CAs in the detection of early thrombus formation. METHODS: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an alpha(2)-antiplasmin-based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of alpha(2)-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. RESULTS: In vitro-generated thrombi exposed to the alpha(2)-antiplasmin-based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the alpha(2)-antiplasmin-based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the alpha(2)-antiplasmin-based CA bound to fibrin. Immunohistochemistry demonstrated substantial alpha(2)-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 +/- 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs -0.14 +/- 0.55, p = 0.003, n = 6) and alpha(2)-antiplasmin-based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 +/- 0.23, p = 0.006, n = 6). CONCLUSIONS: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.
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Medios de Contraste , Gadolinio DTPA , Imagen por Resonancia Magnética , Embolia Pulmonar/diagnóstico , Rodaminas , Trombosis/diagnóstico , alfa 2-Antiplasmina , Animales , Cadaverina/análogos & derivados , Cadaverina/farmacología , Modelos Animales de Enfermedad , Factor XIII/metabolismo , Factor XIIIa/metabolismo , Estudios de Factibilidad , Fibrina/metabolismo , Gadolinio DTPA/análogos & derivados , Gadolinio DTPA/farmacocinética , Humanos , Inmunohistoquímica , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Valor Predictivo de las Pruebas , Embolia Pulmonar/sangre , Embolia Pulmonar/patología , Reproducibilidad de los Resultados , Rodaminas/farmacocinética , Trombosis/sangre , Trombosis/patología , alfa 2-Antiplasmina/análogos & derivados , alfa 2-Antiplasmina/farmacologíaRESUMEN
Chemoselective ligation methods have increased the efficiency of bioconjugation, enabling complex macromolecules to be assembled. In particular, these methods have been utilized for the ligation and modification of peptides and proteins. The chemical synthesis of proteins from unprotected peptide fragments has enabled the introduction of unnatural amino acids, site-specific isotopic labeling, and the site-specific attachment of affinity tags or labels for imaging. A greater insight into current ligation methods has led to higher reaction rates, higher reaction yields, and greater biocompatibility, thereby increasing the impact of chemoselective ligation reactions in chemical biology.
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Péptidos/química , Humanos , Compuestos Orgánicos/química , Fragmentos de Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato , Azufre/químicaRESUMEN
The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature.
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Adenocarcinoma/irrigación sanguínea , Neoplasias Colorrectales/irrigación sanguínea , Medios de Contraste/química , Angiografía por Resonancia Magnética/métodos , Oligopéptidos/química , Puntos Cuánticos , Algoritmos , Animales , Unión Competitiva , Medios de Contraste/farmacocinética , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Masculino , Ratones , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oligopéptidos/farmacocinética , Distribución Tisular , Trasplante HeterólogoRESUMEN
A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.