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1.
Contrast Media Mol Imaging ; 9(3): 211-20, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24700748

RESUMO

Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with (99m) Tc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA(pos) LNCaP and PSMA(neg) PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. (99m) Tc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. (99m) Tc-PSMA30 internalized significantly more in LNCaP cells compared to (99m) Tc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for (99m) Tc-PSMA30 compared with (99m) Tc-PSMA6 and a (99m) Tc-control nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting.


Assuntos
Glutamato Carboxipeptidase II/antagonistas & inibidores , Imagem Molecular/métodos , Neoplasias da Próstata/diagnóstico , Compostos Radiofarmacêuticos , Anticorpos de Domínio Único , Tecnécio , Animais , Antígenos de Superfície/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glutamato Carboxipeptidase II/imunologia , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Células Tumorais Cultivadas , Microtomografia por Raio-X , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Hybridoma (Larchmt) ; 31(6): 424-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23244321

RESUMO

Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of non-prostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs) specific for the external domain of the PSMA are reported. Due to the absence of the CH1 domain, HCAbs are smaller than their counterparts in conventional antibodies. In this study, camel antibodies were generated through immunization of Camelus dromedarius with a synthetic 28 amino acid peptide corresponding to the external surface domain of antigen and PSMA-expressing cell lines. Different binding properties to protein A and protein G affinity columns were deployed to separate three subclasses of camel IgG. The affinity purified HCAbs bound selectively to the synthetic peptide in enzyme linked immunosorbent assay (ELISA) and reacted specifically with PSMA-expressing cell line through immunocytochemistry study. Currently, we are attempting to develop recombinant variable domain of these heavy chain antibodies (VHH or nanobody) for tumor imaging and cancer therapy.


Assuntos
Anticorpos/química , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Animais , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Antígenos de Superfície/metabolismo , Camelus , Linhagem Celular Tumoral , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Glutamato Carboxipeptidase II/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Neoplasias da Próstata , Ligação Proteica , Soroalbumina Bovina/química
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