RESUMO
PURPOSE: Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mAbs) for imaging and therapy. The positron emitter zirconium-89 ((89)Zr) has attractive characteristics for immuno-PET with intact mAbs. Previously, we have described a multi-step procedure for stable coupling of (89)Zr to mAbs via the bifunctional chelate (BFC) tetrafluorophenol-N-succinyldesferal (TFP-N-sucDf). To enable widespread use of (89)Zr-immuno-PET, we now introduce the novel BFC p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) and compare its performance in (89)Zr-immuno-PET with the reference BFC TFP-N-sucDf. METHODS: Three mAbs were premodified with Df-Bz-NCS and labeled with (89)Zr at different pHs to assess the reaction kinetics and robustness of the radiolabeling. Stability of both (89)Zr-Df-Bz-NCS- and (89)Zr-N-sucDf-conjugates was evaluated in different buffers and human serum. Comparative biodistribution and PET studies in tumor-bearing mice were undertaken. RESULTS: The selected conjugation conditions resulted in a chelate:mAb substitution ratio of about 1.5:1. Under optimal radiolabeling conditions (pH between 6.8-7.2), the radiochemical yield was >85% after 60 min incubation at room temperature, resulting in radioimmunoconjugates with preserved integrity and immunoreactivity. The new radioimmunoconjugate was very stable in serum for up to 7 days at 37 degrees C, with <5% (89)Zr release, and was equally stable compared to the reference conjugate when stored in the appropriate buffer at 4 degrees C. In biodistribution and imaging experiments, the novel and the reference radioimmunoconjugates showed high and similar accumulation in tumors in nude mice. CONCLUSIONS: The novel Df-Bz-NCS BFC allows efficient and easy preparation of optimally performing (89)Zr-labeled mAbs, facilitating further exploration of (89)Zr-immuno-PET as an imaging tool.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Carcinoma de Células Escamosas/metabolismo , Quelantes/química , Desferroxamina/análogos & derivados , Isotiocianatos/química , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/farmacocinética , Zircônio/farmacocinética , Animais , Carcinoma de Células Escamosas/diagnóstico por imagem , Linhagem Celular Tumoral , Desferroxamina/química , Feminino , Humanos , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Especificidade de Órgãos , Radioimunodetecção/métodos , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Zircônio/químicaRESUMO
PURPOSE: The human monoclonal antibody (MAb) fragment L19-SIP is directed against extra domain B (ED-B) of fibronectin, a marker of tumour angiogenesis. A clinical radioimmunotherapy (RIT) trial with (131)I-L19-SIP was recently started. In the present study, after GMP production of (124)I and efficient production of (124)I-L19-SIP, we aimed to demonstrate the suitability of (124)I-L19-SIP immuno-PET for imaging of angiogenesis at early-stage tumour development and as a scouting procedure prior to clinical (131)I-L19-SIP RIT. METHODS: (124)I was produced in a GMP compliant way via (124)Te(p,n)(124)I reaction and using a TERIMO module for radioiodine separation. L19-SIP was radioiodinated by using a modified version of the IODO-GEN method. The biodistribution of coinjected (124)I- and (131)I-L19-SIP was compared in FaDu xenograft-bearing nude mice, while (124)I PET images were obtained from mice with tumours of <50 to approximately 700 mm(3). RESULTS: (124)I was produced highly pure with an average yield of 15.4 +/- 0.5 MBq/microAh, while separation yield was approximately 90% efficient with <0.5% loss of TeO(2). Overall labelling efficiency, radiochemical purity and immunoreactive fraction were for (124)I-L19-SIP: approximately 80 , 99.9 and >90%, respectively. Tumour uptake was 7.3 +/- 2.1, 10.8 +/- 1.5, 7.8 +/- 1.4, 5.3 +/- 0.6 and 3.1 +/- 0.4%ID/g at 3, 6, 24, 48 and 72 h p.i., resulting in increased tumour to blood ratios ranging from 6.0 at 24 h to 45.9 at 72 h p.i.. Fully concordant labelling and biodistribution results were obtained with (124)I- and (131)I-L19-SIP. Immuno-PET with (124)I-L19-SIP using a high-resolution research tomograph PET scanner revealed clear delineation of the tumours as small as 50 mm(3) and no adverse uptake in other organs. CONCLUSIONS: (124)I-MAb conjugates for clinical immuno-PET can be efficiently produced. Immuno-PET with (124)I-L19-SIP appeared qualified for sensitive imaging of tumour neovasculature and for predicting (131)I-L19-SIP biodistribution.
Assuntos
Anticorpos/uso terapêutico , Radioisótopos do Iodo , Neoplasias/irrigação sanguínea , Neoplasias/radioterapia , Neovascularização Patológica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Radioimunoterapia/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos/administração & dosagem , Anticorpos/química , Linhagem Celular Tumoral , Estudos de Viabilidade , Humanos , Injeções , Radioisótopos do Iodo/farmacocinética , Marcação por Isótopo , Camundongos , Estadiamento de Neoplasias , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Planejamento da Radioterapia Assistida por Computador , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Distribuição Tecidual , Transplante Heterólogo , Ureia/análogos & derivados , Ureia/químicaRESUMO
The approximately 15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti-epidermal growth factor receptor (EGFR) Nanobody (alphaEGFR-alphaEGFR) by fusion to a Nanobody unit binding to albumin (alphaAlb). Biodistributions of alphaEGFR-alphaEGFR, alphaEGFR-alphaEGFR-alphaAlb ( approximately 50 kDa), alphaTNF-alphaTNF-alphaAlb (control, binding tumor necrosis factor-alpha), and the approximately 150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with (177)Lu to facilitate quantification. Tumor uptake of (177)Lu-alphaEGFR-alphaEGFR decreased from 5.0 +/- 1.4 to 1.1 +/- 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of alphaAlb. Blood levels of alphaEGFR-alphaEGFR-alphaAlb were 21.2 +/- 2.5, 11.9 +/- 0.6, and 4.0 +/- 1.4 and tumor levels were 19.4 +/- 5.5, 35.2 +/- 7.5, and 28.0 +/- 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 +/- 3.9, 27.1 +/- 7.9, and 25.6 +/- 6.1 %ID/g) and significantly higher than for alphaTNF-alphaTNF-alphaAlb. alphaEGFR-alphaEGFR-alphaAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of alphaEGFR and alphaAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned.
Assuntos
Albuminas/metabolismo , Anticorpos/imunologia , Receptores ErbB/imunologia , Nanotecnologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Cetuximab , Receptores ErbB/metabolismo , Ligação Proteica , Distribuição TecidualRESUMO
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.
Assuntos
Apoptose/efeitos dos fármacos , Microdomínios da Membrana/química , Éteres Fosfolipídicos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/biossíntese , Androstenos/farmacologia , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Colesterol/análise , Colesterol/metabolismo , Regulação para Baixo , Endocitose/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Expressão Gênica/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Norbornanos , Éteres Fosfolipídicos/metabolismo , RNA Interferente Pequeno/farmacologia , Esfingomielinas/análise , Esfingomielinas/metabolismo , Tiocarbamatos , Tionas/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Células Tumorais CultivadasRESUMO
UNLABELLED: The extra domain B of fibronectin (ED-B) is a marker of tumor angiogenesis. The human monoclonal antibody (mAb) L19-SIP (approximately 80 kDa; SIP is "small immunoprotein") has been selected for targeting of ED-B. The aim of this study was to evaluate the potential of radioimmunotherapy (RIT) with L19-SIP, either alone or in combination with cetuximab, for treatment of head and neck squamous cell carcinoma (HNSCC). Combination with cetuximab was considered because this anti-EGFR (epidermal growth factor receptor) mAb has proven value for the treatment of HNSCC. METHODS: HNSCC xenograft lines FaDu and HNX-OE were evaluated for ED-B and EGFR expression. L19-SIP was radiolabeled with 2 candidate radionuclides for RIT, 177Lu and 131I (or 125I as substitute). The biodistribution of coinjected 177Lu-L19-SIP and 125I-L19-SIP was assessed in FaDu-bearing nude mice, whereas 131I-L19-SIP was evaluated in both xenograft lines. After labeling with high-dose 131I (623-789 MBq/mg), the maximum tolerated dose (MTD) was assessed. The efficacy of RIT with injected 131I-L19-SIP, either alone or in combination with unlabeled cetuximab (1 mg 2 times a week intraperitoneally for 4 wk), was evaluated in both xenograft lines. RESULTS: Xenograft lines expressed both antigens, with similar EGFR expression and the highest ED-B expression in FaDu. Radioiodinated L19-SIP performed better than 177Lu-L19-SIP and was further exploited. The biodistribution of 131I-L19-SIP was most favorable in FaDu-bearing mice, with tumor uptake values at 24, 48, and 72 h after injection of 8.6 +/- 1.6, 5.8 +/- 0.4, and 3.4 +/- 0.2 %ID/g (%ID/g is percentage injected dose per gram of tissue), respectively, and ratios of tumor to normal tissues that gradually increased in time, such as for blood from 4.4 +/- 1.8 at 24 h to 21.4 +/- 1.7 at 72 h, after injection. RIT at the MTD level of 74 MBq caused significant tumor growth delay and improved survival in both lines. Although FaDu was most sensitive for RIT, with size reduction of all tumors, HNX-OE was most sensitive for treatment with cetuximab. The best survival and cure rates were obtained, however, when RIT and cetuximab were combined. CONCLUSION: RIT with 131I-L19-SIP appeared efficacious in HNSCC xenografts. The efficacy of RIT was enhanced by combination with cetuximab, without increase of toxicity.
Assuntos
Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Radioisótopos do Iodo/uso terapêutico , Neoplasias/patologia , Neovascularização Patológica , Radioimunoterapia/métodos , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Estrutura Terciária de ProteínaRESUMO
The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541-39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.
Assuntos
Apoptose , Endocitose , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/química , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Compostos de Boro/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Dinaminas/metabolismo , Dinaminas/fisiologia , Citometria de Fluxo/métodos , Células HeLa , Humanos , Lactosilceramidas/metabolismo , Lisofosfatidilcolinas/química , Lisofosfolipídeos/antagonistas & inibidores , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Transfecção , Trítio , Células Tumorais CultivadasRESUMO
The positron emitter zirconium-89 ((89)Zr) has very attractive properties for positron emission tomography (PET) imaging of intact monoclonal antibodies (mAbs) using immuno-PET. This protocol describes the step-by-step procedure for the facile radiolabeling of mAbs or other proteins with (89)Zr using p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). First, Df-Bz-NCS is coupled to the lysine-NH(2) groups of a mAb at pH 9.0 (pre-modification), followed by purification using gel filtration. Next, the pre-modified mAb is labeled at room temperature by the addition of [(89)Zr]Zr-oxalic acid solution followed by purification using gel filtration. The entire process of pre-modification, radiolabeling and purification steps will take about 2.5 h.
Assuntos
Anticorpos Monoclonais , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/síntese química , Zircônio , Animais , Anticorpos Monoclonais/química , Quelantes , Desferroxamina/análogos & derivados , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Humanos , Isotiocianatos , Camundongos , Camundongos Nus , Radioisótopos , Compostos Radiofarmacêuticos/química , Transplante HeterólogoRESUMO
Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fosfolipídeos/farmacologia , Alquilação , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/crescimento & desenvolvimento , Humanos , Indicadores e Reagentes , Inibidores de Fosfodiesterase/farmacologia , Éteres Fosfolipídicos/farmacologia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologiaRESUMO
The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49(AR). ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49(AR) cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49(AR) cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-beta-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.