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1.
Sci Adv ; 6(6): eaav7504, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32083172

RESUMO

Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (r 1 = 30.9 mM-1 s-1, r 2 = 43.2 mM-1 s-1, 1.5 T; r 1 = 23.5 mM-1 s-1, r 2 = 98.6 mM-1 s-1, 7.0 T), strong CXCR4 binding (K d = 1.10 ± 0.18 µM), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm3. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.


Assuntos
Biomarcadores , Meios de Contraste , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/metabolismo , Imagem por Ressonância Magnética , Imagem Molecular , Receptores CXCR4/metabolismo , Animais , Meios de Contraste/química , Modelos Animais de Doenças , Detecção Precoce de Câncer , Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Imagem por Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Metástase Neoplásica , Ligação Proteica , Curva ROC , Receptores CXCR4/química , Receptores CXCR4/genética , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
2.
Nat Commun ; 10(1): 4777, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664017

RESUMO

Early diagnosis and noninvasive detection of liver fibrosis and its heterogeneity remain as major unmet medical needs for stopping further disease progression toward severe clinical consequences. Here we report a collagen type I targeting protein-based contrast agent (ProCA32.collagen1) with strong collagen I affinity. ProCA32.collagen1 possesses high relaxivities per particle (r1 and r2) at both 1.4 and 7.0 T, which enables the robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis and nonalcoholic steatohepatitis (Ishak stage 1 of 6 or 1 A Mild) in animal models via dual contrast modes. ProCA32.collagen1 also demonstrates vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and heterogeneity via serial molecular imaging. ProCA32.collagen1 mitigates metal toxicity due to lower dosage and strong resistance to transmetallation and unprecedented metal selectivity for Gd3+ over physiological metal ions with strong translational potential in facilitating effective treatment to halt further chronic liver disease progression.


Assuntos
Meios de Contraste/química , Gadolínio/química , Hipertensão Portal/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Doença Crônica , Diagnóstico Precoce , Humanos
3.
Biomaterials ; 224: 119478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31542517

RESUMO

The Liver is the most common organ for metastasis for various cancers, including uveal melanoma, the most common primary intraocular tumor. Uveal melanoma metastasizes to the liver in ~90% of patients, and results in death in almost all cases due to late detection and lack of effective treatment. There is a pressing unmet medical need to develop MRI contrast agents and imaging methodologies with desired sensitivity and specificity to overcome the high heterogeneous background and in vivo properties as well as reduced toxicity. Herein, we report the development of a collagen targeting protein contrast agent (ProCA32.collagen1), since collagen is a diagnostic biomarker and therapeutic target for many types of primary and metastatic cancers and the tumor microenvironment. In addition to a strong affinity to collagen I, ProCA32.collagen1 possesses high relaxivities (r1 and r2 are 68.0 ±â€¯0.25 and 100.0 ±â€¯0.32 mM-1 s-1 at 1.4 T, respectively, and 42.6 ±â€¯1.0 and 217 ±â€¯2.4 mM-1s-1 at 7.0 T per particle). ProCA32.collagen1 also has strong serum stability against degradation, resistance to transmetallation, and 102 and 1013-fold higher metal selectivity for Gd3+ over Ca2+ and Zn2+, respectively, compared to clinical contrast agents. ProCA32.collagen1 does not exhibit any cell toxicity for various cell lines. Sensitive detection of liver lesions in animal models can be achieved using multiple imaging methodologies, taking advantage of the dual relaxation property of ProCA32.collagen1. ProCA32.collagen1 enables sensitive and early stage detection of hepatic micrometastasis as small as 0.144 mm2 and two different tumor growth patterns. Further development of ProCA32.collagen1 has the potential to greatly facilitate non-invasive, early detection and staging of primary and metastatic liver cancers, and devising effective treatments.


Assuntos
Colágeno/química , Meios de Contraste/química , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Imagem por Ressonância Magnética , Animais , Linhagem Celular , Sobrevivência Celular , Endocitose , Feminino , Humanos , Fígado/patologia , Camundongos Endogâmicos C57BL , Distribuição Tecidual
4.
J Agric Food Chem ; 67(32): 8746-8755, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31322881

RESUMO

The underlying mechanisms of the higher photosynthetic efficiency of cultivated cassava relative to its wild species are poorly understood. In the present study, proteins in leaves and chloroplasts were analyzed to compare the differences among the cultivar SC205, its wild ancestor W14, and the related species Glaziovii. The functions of differential proteins are associated with 10 ontology groups including photosynthesis, carbohydrate and energy metabolism, as well as potential signal pathway. The protein-protein networks among 41 differential proteins showed that PGK1 is a hub protein and protein cross-interactions affected the differentiation of photosynthetic rate. Anatomy patterns and PEPC detection suggested that SC205 has more C4 photosynthesis characteristics than Glaziovii and W14. Finally, a mechanism model of the efficient photosynthesis was proposed based on the remarkable variations in photosynthetic parameters and protein functions in the domestic cultivars.


Assuntos
Manihot/metabolismo , Fotossíntese , Cloroplastos/metabolismo , Manihot/classificação , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas
5.
Nanoscale ; 8(25): 12668-82, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26961235

RESUMO

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd(3+) contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd(3+) binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 µM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10(-22) M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM(-1) s(-1) and r2 of 37.9 mM(-1) s(-1) per Gd (55.2 and 75.8 mM(-1) s(-1) per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM(-1) s(-1) per Gd (188.0 mM(-1) s(-1) per molecule) and r1 of 18.6 mM(-1) s(-1) per Gd (37.2 mM(-1) s(-1) per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.


Assuntos
Antígenos de Superfície/análise , Meios de Contraste , Glutamato Carboxipeptidase II/análise , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Gadolínio , Humanos , Masculino , Camundongos , Camundongos Nus
6.
Curr Protein Pept Sci ; 17(6): 519-33, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721404

RESUMO

Prostate cancer is the most common cancer for man with a high mortality rate due to a lack of non-invasive accurate and sensitive molecular diagnostic methods. The molecular imaging of cancer biomarkers using MRI with its spatial and temporal resolution, however, is largely limited by the lack of contrast agents with high sensitivity, targeting specificity and deep tumor penetration. In this review, we will first overview the current stage of prostate cancer diagnosis and then review prostate cancer biomarkers and related imaging techniques. Since biomarker targeting moieties are essential for molecular imaging, we will use prostate-specific membrane antigen (PSMA) as an example to discuss different methods to characterize the interaction between biomarker and targeting moieties. At the end, we will review current progress of the development of targeted protein-based MRI contrast agents (ProCAs) for prostate cancer biomarkers with improved relaxivity and targeting capability.


Assuntos
Biomarcadores Tumorais , Meios de Contraste , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Diagnóstico por Imagem/métodos , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Masculino , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Ligação Proteica , Proteínas/química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptores da Bombesina/química , Receptores da Bombesina/metabolismo
7.
Sci Rep ; 5: 16214, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26577829

RESUMO

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd(3+) binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM(-1)s(-1) at 1.5 T and 25 °C) and strong Gd(3+) selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.


Assuntos
Meios de Contraste , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptores da Bombesina/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/metabolismo , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Expressão Gênica , Xenoenxertos , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/métodos , Neoplasias/genética , Ligação Proteica , Ratos , Receptores da Bombesina/química , Receptores da Bombesina/genética , Distribuição Tecidual
8.
Proc Natl Acad Sci U S A ; 112(21): 6607-12, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25971726

RESUMO

With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd(3+) toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd(3+) and a 10(11)-fold greater selectivity for Gd(3+) over Zn(2+) compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol(-1)⋅s(-1)/89.2 mmol(-1)⋅s(-1) per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10-20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.


Assuntos
Meios de Contraste , Neoplasias Hepáticas Experimentais/diagnóstico , Neoplasias Hepáticas Experimentais/metabolismo , Imagem por Ressonância Magnética/métodos , Melanoma Experimental/diagnóstico , Melanoma Experimental/metabolismo , Parvalbuminas , Animais , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/farmacocinética , Feminino , Gadolínio , Limite de Detecção , Neoplasias Hepáticas Experimentais/secundário , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parvalbuminas/química , Parvalbuminas/farmacocinética , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética
9.
J Biol Chem ; 289(37): 25812-21, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25070887

RESUMO

It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.


Assuntos
Proteínas de Transporte/sangue , Proliferação de Células/genética , Proteínas de Membrana/sangue , Neovascularização Patológica/patologia , Isoformas de Proteínas/sangue , Hormônios Tireóideos/sangue , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Glicólise/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neoplasias/sangue , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Neovascularização Patológica/sangue , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Med Res Rev ; 34(5): 1070-99, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24615853

RESUMO

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three-dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein-based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein-based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal-binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.


Assuntos
Biomarcadores Tumorais/análise , Meios de Contraste , Gadolínio/administração & dosagem , Imagem por Ressonância Magnética/métodos , Relação Dose-Resposta a Droga , Gadolínio/química
11.
J Biol Inorg Chem ; 19(2): 259-70, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24366655

RESUMO

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r1 and r2 relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.


Assuntos
Meios de Contraste , Receptores ErbB/metabolismo , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/patologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-23335551

RESUMO

Magnetic resonance imaging (MRI) of disease biomarkers, especially cancer biomarkers, could potentially improve our understanding of the disease and drug activity during preclinical and clinical drug treatment and patient stratification. MRI contrast agents with high relaxivity and targeting capability to tumor biomarkers are highly required. Extensive work has been done to develop MRI contrast agents. However, only a few limited literatures report that protein residues can function as ligands to bind Gd(3+) with high binding affinity, selectivity, and relaxivity. In this paper, we focus on reporting our current progress on designing a novel class of protein-based Gd(3+) MRI contrast agents (ProCAs) equipped with several desirable capabilities for in vivo application of MRI of tumor biomarkers. We will first discuss our strategy for improving the relaxivity by a novel protein-based design. We then discuss the effect of increased relaxivity of ProCAs on improving the detection limits for MRI contrast agent, especially for in vivo application. We will further report our efforts to improve in vivo imaging capability and our achievement in molecular imaging of cancer biomarkers with potential preclinical and clinical applications.


Assuntos
Biomarcadores Tumorais/análise , Meios de Contraste/química , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Proteínas/química , Animais , Linhagem Celular Tumoral , Humanos , Limite de Detecção
13.
J Inorg Biochem ; 107(1): 111-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22178673

RESUMO

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd(3+) binding sites into a stable protein resulting in significantly increased longitudinal (r(1)) and transverse (r(2)) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r(1) and r(2) relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.


Assuntos
Proteínas de Transporte/química , Meios de Contraste/síntese química , Complexos de Coordenação/química , Polietilenoglicóis/química , Animais , Sítios de Ligação , Bioengenharia , Proteínas de Transporte/efeitos adversos , Proteínas de Transporte/farmacocinética , Linhagem Celular Tumoral , Meios de Contraste/efeitos adversos , Meios de Contraste/farmacocinética , Complexos de Coordenação/efeitos adversos , Complexos de Coordenação/farmacocinética , Gadolínio/química , Gadolínio DTPA/química , Humanos , Imagem por Ressonância Magnética , Teste de Materiais , Camundongos , Modelos Moleculares , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/farmacocinética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Solubilidade
14.
PLoS One ; 6(3): e18103, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21455310

RESUMO

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.


Assuntos
Meios de Contraste/química , Imagem por Ressonância Magnética/métodos , Receptor ErbB-2/imunologia , Animais , Anticorpos/química , Anticorpos/imunologia , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
15.
Mol Imaging Biol ; 13(3): 416-423, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20574851

RESUMO

PURPOSE: The purpose of this study was to demonstrate a novel protein-based magnetic resonance imaging (MRI) contrast agent that has the capability of targeting prostate cancer and which provides high-sensitivity MR imaging in tumor cells and mouse models. PROCEDURE: A fragment of gastrin-releasing peptide (GRP) was fused into a protein-based MRI contrast agent (ProCA1) at different regions. MR imaging was obtained in both tumor cells (PC3 and H441) and a tumor mouse model administrated with ProCA1.GRP. RESULTS: PC3 and DU145 cells treated with ProCA1.GRPs exhibited enhanced signal in MRI. Intratumoral injection of ProCA1.GRP in a PC3 tumor model displayed enhanced MRI signal. The contrast agent was retained in the PC3 tumor up to 48 h post-injection. CONCLUSIONS: Protein-based MRI contrast agent with tumor targeting modality can specifically target GRPR-positive prostate cancer. Intratumoral injection of the ProCA1 agent in the prostate cancer mouse model verified the targeting capability of ProCA1.GRP and showed a prolonged retention time in tumors.


Assuntos
Proteínas de Transporte , Meios de Contraste , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Neoplasias da Próstata/diagnóstico , Animais , Meios de Contraste/química , Peptídeo Liberador de Gastrina , Masculino , Camundongos , Fatores de Tempo
16.
Protein Expr Purif ; 47(1): 249-57, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16403645

RESUMO

Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Clonagem Molecular , Códon/biossíntese , Pichia , Receptor ErbB-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Códon/química , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Dados de Sequência Molecular , Pichia/genética
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