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1.
Proc Natl Acad Sci U S A ; 111(48): 17110-5, 2014 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-25406323

RESUMEN

Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased half-life, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.


Asunto(s)
Proteínas Portadoras/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Unión Competitiva , Proteínas Portadoras/genética , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Semivida , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones Endogámicos , Biblioteca de Péptidos , Unión Proteica , Receptores Fc/genética , Proteínas Recombinantes de Fusión/sangre
2.
Cancer Res ; 67(5): 2178-86, 2007 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-17332348

RESUMEN

The Affibody molecule Z(HER2:342-pep2), site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z(HER2:342-pep2) folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of (111)In was achieved within 30 min at low (room temperature) and high temperatures (up to 90 degrees C). Tumor uptake of (111)In-DOTA-Z(HER2:342-pep2) was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of (111)In-DOTA-Z(HER2:342-pep2) obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z(HER2:342-pep2) has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.


Asunto(s)
Adenocarcinoma/diagnóstico , Diagnóstico por Imagen/métodos , Compuestos Organometálicos , Neoplasias Ováricas/diagnóstico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Afinidad de Anticuerpos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Técnicas de Diagnóstico Molecular/métodos , Compuestos Organometálicos/farmacocinética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/farmacocinética , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Biochem Biophys Res Commun ; 377(2): 489-494, 2008 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-18930032

RESUMEN

HER2-specific affibody molecules in different formats have previously been shown to be useful tumor targeting agents for radionuclide-based imaging and therapy applications, but their biological effect on tumor cells is not well known. In this study, two dimeric ((Z(HER2:4))(2) and (Z(HER2:342))(2)) and one monomeric (Z(HER2:342)) HER2-specific affibody molecules are investigated with respect to biological activity. Both (Z(HER2:4))(2) and (Z(HER2:342))(2) were found to decrease the growth rate of SKBR-3 cells to the same extent as the antibody trastuzumab. When the substances were removed, the cells treated with the dimeric affibody molecules continued to be growth suppressed while the cells treated with trastuzumab immediately resumed normal proliferation. The effects of Z(HER2:342) were minor on both proliferation and cell signaling. The dimeric (Z(HER2:4))(2) and (Z(HER2:342))(2) both reduced growth of SKBR-3 cells and may prove therapeutically useful either by themselves or as carriers of radionuclides or other cytotoxic agents.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Receptor ErbB-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Técnicas Biosensibles , Línea Celular , Línea Celular Tumoral , Dimerización , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Trastuzumab
4.
PLoS One ; 8(11): e81350, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24260574

RESUMEN

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.


Asunto(s)
Expresión Génica , Vectores Genéticos/química , Antígenos de Histocompatibilidad Clase I/genética , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Receptores Fc/genética , Proteínas Recombinantes de Fusión/biosíntesis , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Línea Celular Tumoral , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/genética , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Recién Nacido , Lentivirus/genética , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Fc/química , Receptores Fc/inmunología , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica/química , Albúmina Sérica/inmunología , Solubilidad
5.
Protein Eng Des Sel ; 26(10): 635-44, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23924760

RESUMEN

Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K(D), of ~20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)2htt dimer binds with an off-rate of k(off) ≤ 5 × 10(-6) s(-1) and an estimated K(D) ≤ 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.


Asunto(s)
Ingeniería de Proteínas , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína Estafilocócica A/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética
6.
J Nucl Med ; 54(6): 961-8, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23528382

RESUMEN

UNLABELLED: Because of their better penetration, smaller targeting proteins may be superior to antibodies for radioimmunotherapy of solid tumors. Therefore, Affibody molecules (6.5 kDa) have a potential for being suitable as targeted moiety for radiolabeled therapeutic proteins. Previous studies have demonstrated that a fusion of an Affibody molecule with an albumin-binding domain (ABD) provides a strong noncovalent binding to albumin in vivo. This strong noncovalent binding can be used for reduction of the renal uptake of the Affibody molecule while maintaining a size smaller than that of an antibody, which is important when using residualizing radionuclide labels conjugated to Affibody molecules. The goal of this study was to design and evaluate a new targeting Affibody-ABD fusion protein with improved biodistribution properties for radionuclide therapy. METHODS: A novel Affibody-based construct, ZHER2:2891-ABD035-DOTA (ABY-027), was created by fusion of the reengineered HER2-binding Affibody molecule ZHER2:2891 to the N terminus of the high-affinity ABD035, and a maleimido-derivative of DOTA was conjugated at the C terminus of the construct. Binding and processing of (177)Lu-ABY-027 by HER2-expressing cells were evaluated in vitro. Targeting of HER2-expressing SKOV-3 xenografts was evaluated in BALB/C nu/nu mice and compared with targeting of previously reported ABD-(ZHER2:342)2. RESULTS: The binding affinity (dissociation constant) of ABY-027 to HER2 (74 pM) was the same as for the parental ZHER2:2891 (76 pM). ABY-027 was stably labeled with (177)Lu and (111)In with preserved specific binding to HER2-expressing cells in vitro. In vivo receptor saturation experiments demonstrated that targeting of SKOV-3 xenografts in BALB/C nu/nu mice was HER2-specific. (177)Lu-ABY-027 demonstrated substantially (2- to 3-fold) lower renal and hepatic uptake than previously assessed HER2-specific Affibody-based albumin-binding agents. Tumor uptake of radiolabeled ABY-027 at 48 h after injection was 2-fold higher than that for previously reported ABD-(ZHER2:342)2. CONCLUSION: An optimized molecular design of an ABD fusion protein resulted in an Affibody molecule construct with better properties for therapy. Fully preserved in vivo targeting of the fusion protein was shown in xenografted mice. Site-specific coupling of DOTA provides a uniform conjugate and creates the potential for labeling with a broad range of therapeutic radionuclides. The biodistribution of (177)Lu-ABY-027 in a murine model suggests it is more suitable for therapy than alternative approaches.


Asunto(s)
Albúminas/metabolismo , Lutecio/uso terapéutico , Radioisótopos/uso terapéutico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Sitios de Unión , Línea Celular Tumoral , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Marcaje Isotópico , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/radioterapia , Estructura Terciaria de Proteína , Transporte de Proteínas , Radiometría , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Especificidad por Sustrato , Distribución Tisular
7.
Nucl Med Biol ; 38(5): 697-706, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21718945

RESUMEN

INTRODUCTION: Affibody molecules are small (∼6.5 kDa) scaffold proteins suitable for radionuclide imaging of tumor-associated molecular targets. Site-specific labeling of Affibody molecules made by peptide synthesis can be achieved by coupling a chelator to N-terminus in the last synthesis step. The goal of this study was to evaluate the influence of a 6-aminohexanoic linker between DOTA and Z(HER2:342) on targeting properties of (111)In-labeled conjugate. METHODS: A DOTA-conjugated 6-aminohexanoic linker-containing variant of Z(HER2:342) (ABY-003) was produced by peptide synthesis, and the in vitro binding affinity, specificity and cellular processing were evaluated. The biodistribution of (111)In-ABY-003 in normal mice was compared to (111)In-ABY-002 (DOTA-Z(HER2:342-pep2)) lacking the linker. Tumor-targeting properties of (111)In-ABY-003 were evaluated in mice bearing HER2-expressing xenografts. RESULTS: The dissociation constant of ABY-003 was in the low picomolar range, slightly higher than for ABY-002. (111)In-ABY-003 bound specifically to HER2-expressing cells in vitro. The cellular retention was efficient but slightly worse than for (111)In-ABY-002. In normal mice, the clearance of (111)In-ABY-003 from blood and other tissues was slightly but significantly faster compared to (111)In-ABY-002. Targeting of HER2-expressing xenografts by (111)In-ABY-003 was receptor-specific. Due to faster clearance, the tumor-to-blood ratio for (111)In-ABY-003 at 4 h postinjection was improved compared to (111)In-ABY-002. The capacity of (111)In-ABY-003 to visualize HER2-expressing tumors was confirmed by gamma camera imaging. CONCLUSIONS: A 6-aminohexanoic linker between the DOTA chelator and N-terminus of synthetic Z(HER2:342) had a measurable effect on affinity, cellular retention of radioactivity and blood clearance. The linker might be used for modulation of targeting properties of Affibody molecules.


Asunto(s)
Compuestos Heterocíclicos con 1 Anillo/química , Radioisótopos de Indio , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Ácido Aminocaproico/química , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Marcaje Isotópico , Ratones , Datos de Secuencia Molecular , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Péptidos/química , Cintigrafía , Proteínas Recombinantes de Fusión/farmacocinética
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