RESUMO
Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong ß emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/imunologia , Radioisótopos de Ítrio/química , Adulto , Idoso , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Daclizumabe , Feminino , Doença de Hodgkin/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Recidiva , Adulto JovemRESUMO
Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent <2% of cells in lymphoid tissues (LTs). Using a combination of single-photon emission computed tomography imaging and a radiotracer ((99m)Tc-labeled rhesus immunoglobulin G1 anti-CD4R1 (Fab')2), we sequentially imaged CD4(+) cell recovery in rhesus macaques following total body irradiation (TBI) and reinfusion of vector-transduced, autologous CD34(+) cells. Our results present for the first time a sequential, real-time, noninvasive method to evaluate CD4(+) cell recovery. Importantly, despite myeloablation of circulating leukocytes following TBI, total depletion of CD4(+) lymphocytes in LTs such as the spleen is not achieved. The impact of TBI on LTs and PBLs is discordant, in which as few as 32.4% of CD4(+) cells were depleted from the spleen. In addition, despite full lymphocyte recovery in the spleen and PB, lymph nodes have suboptimal recovery. This highlights concerns about residual disease, endogenous contributions to recovery, and residual LT damage following ionizing irradiation. Such methodologies also have direct application to immunosuppressive therapy and other immunosuppressive disorders, such as those associated with viral monitoring.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Tecido Linfoide/fisiologia , Tomografia Computadorizada de Emissão de Fóton Único , Condicionamento Pré-Transplante , Animais , Medula Óssea/efeitos da radiação , Antígenos CD4/genética , Contagem de Linfócito CD4 , Sistemas Computacionais , Genes Reporter , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Imunoglobulina G/genética , Lentivirus/genética , Linfonodos/imunologia , Linfonodos/efeitos da radiação , Tecido Linfoide/diagnóstico por imagem , Tecido Linfoide/efeitos da radiação , Macaca mulatta , Imagem Multimodal , Especificidade de Órgãos , Quimera por Radiação , Baço/imunologia , Baço/efeitos da radiação , Tomografia Computadorizada por Raios X , Transdução Genética , Transplante Autólogo , Irradiação Corporal TotalRESUMO
Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas/imunologia , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Neoplasias Hepáticas/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoterapia , Neoplasias Hepáticas/imunologia , CamundongosRESUMO
Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior tissue penetration and lower autofluorescence. We recently accessed a new class of readily synthesized NIR cyanines containing a novel C4'-O-alkyl linker, which provides both high chemical stability and excellent optical properties. In this study, we provide the first in vivo analysis of this new class of compounds, represented by the tetrasulfonate FNIR-774 (Frederick NIR 774). Monoclonal antibody (mAb) conjugates of FNIR-774 were compared to conjugates of the commercially available dye (IRDye800CW (IR800)), one of the most widely used NIR fluorophores for clinical translation. Both dyes were conjugated to panitumumab (pan) or cetuximab (cet) with ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. In contrast, in vivo imaging in mice showed different pharmacokinetics between pan-FNIR-774 (1:5) and pan-IR800 (1:5), or cet-FNIR-774 (1:5) and cet-IR800 (1:5). Particularly at the higher labeling density, mAb-FNIR-774 conjugates showed superior specific accumulation in tumors compared with mAb-IR800 conjugates. Thus, FNIR-774 conjugates showed superior in vivo pharmacokinetics compared with IR800 conjugates, independent of the mAb. These results suggest that FNIR-774 is a promising fluorescent probe for NIR optical imaging.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias da Mama/patologia , Carbocianinas/química , Cetuximab/metabolismo , Corantes Fluorescentes/farmacocinética , Alquilação , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Células 3T3 BALB , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Células Cultivadas , Cetuximab/química , Feminino , Citometria de Fluxo , Corantes Fluorescentes/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Panitumumabe , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição TecidualRESUMO
Porphyrin-lipid nanovesicles (PLN) have been developed with intrinsic capabilities as activatable multimodal photonic contrast agents. Radiolabeling of PLN encapsulating drugs could eventually be able to provide quantitative in vivo information for diagnosing and treating diseases. In this study, we developed (99m)Tc-labeled porphyrin-lipid nanovesicles ((99m)Tc-PLN) as a cargo-encapsulated formulation without significant impact on liposome integrity and encapsulation stability. 50 mM calcein was encapsulated into PLN by probe sonication. The size of the PLN was about 150 nm. The PLN were then reacted with (99m)Tc using SnCl2 dissolved in 1 mM HCl as a reducing agent and incubated for 10 min at 22 °C. The radiolabeling efficiency and stability of (99m)Tc-PLN were evaluated by instant thin-layer chromatography and low-pressure liquid chromatography (LPLC). (99m)Tc labeling was successful with a >92% labeling efficiency. LPLC showed that the liposomal elution peaks of the porphyrin-lipid and the calcein overlapped with the radioactivity elution peak of (99m)Tc-labeled PLN. The (99m)Tc-labeling procedure did not change the size of PLN. Encapsulated calcein remained inert inside PLN. Thus, this work lays out a simple and effective radiolabeling method using SnCl2 in HCl in the preparation of (99m)Tc-PLN.
Assuntos
Lipídeos/química , Nanoestruturas/química , Porfirinas/química , Tecnécio/químicaRESUMO
Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced â¼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.
Assuntos
Anticorpos Monoclonais Humanizados , Proteínas de Neoplasias/imunologia , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Receptores de Superfície Celular/imunologia , Zircônio , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacocinética , Western Blotting , Desferroxamina/administração & dosagem , Desferroxamina/química , Feminino , Humanos , Imunoprecipitação , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos , Imagem Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Compostos Radiofarmacêuticos/farmacocinética , Receptores de Superfície Celular/antagonistas & inibidores , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio/farmacocinéticaRESUMO
The radiolabeling of the liposome surface can be a useful tool for in vivo tracking of therapeutic drug loaded liposomes. We investigated radiolabeling therapeutic drug (i.e. an antibiotic, amikacin) loaded liposomes with (99m)Tc, nebulization properties of (99m)Tc-labeled liposomal amikacin for inhalation ((99m)Tc-LAI), and its stability by size exclusion low-pressure liquid chromatography (LPLC). LAI was reacted with (99m)Tc using SnCl2 dissolved in ascorbic acid as a reducing agent for 10 min at room temperature. The labeled products were then purified by anion exchange resin. The purified (99m)Tc-LAI in 1.5% NaCl solution was incubated at 4 °C to assess its stability by LPLC. The purified (99m)Tc-LAI was subjected to studies with a clinically used nebulizer (PARI eFlow®) and the Anderson Cascade Impactor (ACI). The use of ascorbic acid at 0.91 mM resulted in a quantitative labeling efficiency. The LPLC profile showed that the liposomal peak of LAI detected by a UV monitor at both 200 nm and 254 nm overlapped with the radioactivity peak of (99m)Tc-LAI, indicating that (99m)Tc-LAI is suitable for tracing LAI. The ACI study demonstrated that the aerosol droplet size distribution determined gravimetrically was similar to that determined by radioactivity. The liposome surface labeling method using SnCl2 in 0.91 mM ascorbic acid produced (99m)Tc-LAI with a high labeling efficiency and stability that are adequate to evaluate the deposition and clearance of inhaled LAI in the lung by gamma scintigraphy.
Assuntos
Amicacina/administração & dosagem , Antibacterianos/administração & dosagem , Lipossomos , Compostos de Organotecnécio/química , Administração por Inalação , Amicacina/química , Antibacterianos/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Tamanho da Partícula , Espectrofotometria UltravioletaRESUMO
Since the earliest days of the HIV epidemic, the number of CD4(+) T cells per unit volume of blood has been recognized as a major prognostic factor for the development of AIDS in persons with HIV infection. It has also been generally accepted that approximately 2% of total body lymphocytes circulate in the blood. In the present study, we have used a nondepleting humanized anti-CD4 monoclonal antibody labeled with the gamma emitter indium-111 to visualize the CD4(+) T-cell pool in vivo in nonhuman primates with simian HIV infection. A strong correlation was noted between radiotracer uptake in spleen, tonsil, axillary lymph nodes, and peripheral blood CD4 T-cell counts (rho = 0.75, 0.93, and 0.85, respectively, P < .005). The relationship between radiotracer retention in lymphoid tissues and CD4(+) T-cell counts in the circulation was governed by an exponential law. These data provide an estimate for the total number of lymphocytes in the body as being between 1.9 and 2.9 x 10(12) and suggest that the partition between peripheral blood and lymphoid tissue is between 0.3% and 0.5%.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Imunoglobulina G/imunologia , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/radioterapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Especificidade por Substrato , TomografiaRESUMO
Uniform antibody microdistribution throughout tumor nodules is crucial for antibody-targeted therapy, because non-uniform microdistribution leads to suboptimal therapeutic effect, a commonly observed limitation of therapeutic antibodies. Herein, we evaluated the microdistribution of different doses of intraperitoneally injected fluorescence-labeled full-antibody trastuzumab (15, 50, and 150 microg) and its Fab fragment (trastuzumab-Fab: 15 and 50 microg) in a mouse model of ovarian cancer with peritoneal disseminated tumor. A semiquantitative approach (central/peripheral accumulation ratio; C/P ratio) was developed using in situ fluorescence microscopy. Furthermore, we compared the microdistribution of intact trastuzumab with a mixed injection of trastuzumab and trastuzumab-Fab or serial injections of trastuzumab using in situ multicolor fluorescence microscopy. Fluorescence images after the administration of 15 or 50 microg trastuzumab and 15 microg trastuzumab-Fab demonstrated antibody accumulation in the tumor periphery, whereas administration of 150 microg trastuzumab and 50 microg trastuzumab-Fab showed relatively uniform accumulation throughout the tumor nodule. Using serial injections (19-h interval) of trastuzumab-rhodamine green and carboxytetramethylrhodamine (TAMRA), it was observed that the latterly injected trastuzumab-TAMRA was distributed more centrally than trastuzumab-rhodamine green injected first, whereas no difference was observed in the control mixed-injection group. Moreover, the mixed injection of trastuzumab and trastuzumab-Fab showed that trastuzumab-Fab distributed more centrally than the same amount of co-injected trastuzumab. Our results suggest that the strategies of increasing dose and using Fab fragments can be used to achieve a uniform antibody distribution within peritoneal disseminated nodules after intraperitoneal injection. Furthermore, serial-injection and mixed-injection strategies can modify antibody microdistribution within tumors and have the potential for preferential delivery of anticancer drugs to either the tumor periphery or its center.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Peritoneais/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Injeções Intraperitoneais , Camundongos , Microscopia de Fluorescência , Neoplasias Ovarianas/patologia , Peritônio/metabolismo , Distribuição Tecidual , TrastuzumabRESUMO
Background: Despite advances in therapy of Hodgkin's lymphoma (HL), a proportion of patients will not respond or relapse. The authors had previously identified CD25, IL-2Rα, as a target for systemic radioimmunotherapy of HL since most normal cells do not express CD25, but it is expressed by a minority of Hodgkin/Reed-Sternberg (HRS) cells and most Tregs rosetting around HRS cells. Study Design and Treatment: This was a single institution, nonrandomized, open-label phase I/II trial of radiolabeled 90Y-daclizumab, an anti-CD25 monoclonal antibody, BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning treatment followed by autologous hematopoietic stem cell transplant (ASCT). Four patients with refractory and relapsed HL were treated in this trial with 3 patients receiving a single dose of 564.6-574.6 MBq 90Y-daclizumab and the fourth patient receiving two doses of 580.9-566.1 MBq 90Y-daclizumab followed by high-dose chemotherapy and ASCT. Results: All 4 evaluable patients treated with 90Y-daclizumab obtained complete responses (CRs) that are ongoing 4.5-7 years following their stem cell transplant. The spectrum and severity of adverse events were mild and more importantly none of the patients, including several with multiple therapies before this treatment, developed the myelodysplastic syndrome. Discussion: Targeting by daclizumab was not directed primarily at tumor cells, but rather the nonmalignant CD25-expressing T cells adjacent to the HRS cells and 90Y-daclizumab provided strong enough ß emissions to kill CD25-negative tumor cells at a distance by a crossfire effect. Furthermore, the strong ß irradiation killed normal cells in the tumor microenvironment. Conclusions: 90Y-daclizumab (anti-CD25), high-dose BEAM chemotherapy and ASCT was well tolerated and yielded sustained complete remissions in all 4 patients with recurrent HL patients who completed their treatment. Significance: Despite advances, a proportion of patients with HL will not have a CR to their initial treatment, and some with CRs will relapse. They demonstrated that the addition of 90Y-daclizumab into the preconditioning regimen for refractory and relapsed HL patients with high-dose BEAM chemotherapy and ASCT provided sustained CRs in the 4 patients studied. Two of these patients were highly refractory to multiple prior treatments with bulky disease at entry into this study, including 1 patient who never entered a remission and had failed 6 different therapeutic regimens. Despite the small number of patients treated in this study, the sustained clinical benefit in these patients indicates a highly effective treatment. The daclizumab was directed primarily not at HRS cells themselves but toward nonmalignant T cells rosetting around malignant cells. 90Y provided strong ß emissions that killed antigen nonexpressing tumor cells at a distance by a crossfire effect. Furthermore, the strong ß radiation killed normal cells in the tumor microenvironment that nurtured the malignant cells in the lymphomatous mass. The present study supports expanded analysis of 90Y-daclizumab as part of the regimen of ASCT in patients with refractory and relapsed HL.
Assuntos
Carmustina/uso terapêutico , Citarabina/uso terapêutico , Daclizumabe/uso terapêutico , Etoposídeo/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Doença de Hodgkin/tratamento farmacológico , Melfalan/uso terapêutico , Transplante Autólogo/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carmustina/farmacologia , Citarabina/farmacologia , Daclizumabe/farmacologia , Etoposídeo/farmacologia , Feminino , Humanos , Masculino , Melfalan/farmacologiaRESUMO
PURPOSE: Exposing human tumor cells to sublethal doses of external beam radiation up-regulates expression of tumor antigen and accessory molecules, rendering tumor cells more susceptible to killing by antigen-specific CTLs. This study explored the possibility that exposure to palliative doses of a radiopharmaceutical agent could alter the phenotype of tumor cells to render them more susceptible to T cell-mediated killing. EXPERIMENTAL DESIGN: Here, 10 human tumor cell lines (4 prostate, 2 breast, and 4 lung) were exposed to increasing doses of the radiopharmaceutical samarium-153-ethylenediaminetetramethylenephosphonate ((153)Sm-EDTMP) used in cancer patients to treat pain due to bone metastasis. Fluorescence-activated cell sorting analysis and quantitative real-time PCR analysis for expression of five surface molecules and several tumor-associated antigens involved in prostate cancer were done. LNCaP human prostate cancer cells were exposed to (153)Sm-EDTMP and incubated with tumor-associated antigen-specific CTL in a CTL killing assay to determine whether exposure to (153)Sm-EDTMP rendered LNCaP cells more susceptible to T cell-mediated killing. RESULTS: Tumor cells up-regulated the surface molecules Fas (100% of cell lines up-regulated Fas), carcinoembryonic antigen (90%), mucin-1 (60%), MHC class I (50%), and intercellular adhesion molecule-1 (40%) in response to (153)Sm-EDTMP. Quantitative real-time PCR analysis revealed additional up-regulated tumor antigens. Exposure to (153)Sm-EDTMP rendered LNCaP cells more susceptible to killing by CTLs specific for prostate-specific antigen, carcinoembryonic antigen, and mucin-1. CONCLUSIONS: Doses of (153)Sm-EDTMP equivalent to palliative doses delivered to bone alter the phenotype of tumor cells, suggesting that (153)Sm-EDTMP may work synergistically with immunotherapy to increase the susceptibility of tumor cells to CTL killing.
Assuntos
Neoplasias da Mama/terapia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/terapia , Compostos Organometálicos/uso terapêutico , Compostos Organofosforados/uso terapêutico , Neoplasias da Próstata/terapia , Radioisótopos/farmacologia , Samário/farmacologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Separação Celular , Feminino , Humanos , Masculino , Fenótipo , Linfócitos T Citotóxicos/metabolismo , Resultado do TratamentoRESUMO
A target cell-specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in "dequenching." In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures.
Assuntos
Avidina/farmacocinética , Corantes Fluorescentes/farmacocinética , Neoplasias Ovarianas/metabolismo , Rodaminas/farmacocinética , Animais , Avidina/química , Linhagem Celular Tumoral , Detergentes/química , Feminino , Fluorescência , Corantes Fluorescentes/química , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Neoplasias Ovarianas/patologia , Rodaminas/química , Espectrometria de FluorescênciaRESUMO
UNLABELLED: The aim of this study was to determine if pulsed high-intensity focused ultrasound (HIFU) exposures could enhance tumor uptake of (111)In-MX-B3, a murine IgG1kappa monoclonal antibody directed against the Le(y) antigen. METHODS: MX-B3 was labeled with (111)In, purified, and confirmed for its binding to the antigen-positive A431 cell line. Groups of nude mice were inoculated subcutaneously with A431 tumor cells on both hind flanks. A tumor on one flank was treated with pulsed-HIFU; the other tumor was used as an untreated control. Within 10 min after the HIFU exposure, the mice received intravenous (111)In-MX-B3 for imaging and biodistribution studies. Mice were euthanized at 1, 24, 48, and 120 h after injection for biodistribution studies. RESULTS: The HIFU exposure shortened the peak tumor uptake time (24 vs. 48 h for the control) and increased the peak tumor uptake value (38 vs. 25 %ID/g [percentage injected dose per gram] for the control). The HIFU effect on enhancing tumor uptake was greater at earlier times up to 24 h, but the effect was gradually diminished thereafter. The HIFU effect on enhancing tumor uptake was substantiated by nuclear imaging studies. HIFU also increased the uptake of the antibody in surrounding tissues, but the net increase was marginal compared with the increase in tumor uptake. CONCLUSION: This study demonstrates that pulsed-HIFU significantly enhances the delivery of (111)In-MX-B3 in human epidermoid tumors xenografted in nude mice. The results of this pilot study warrant further evaluation of other treatment regimens, such as repeated HIFU exposures for greater delivery enhancement of antibodies labeled with cytotoxic radioisotopes or pulsed-HIFU exposure in addition to a combined therapy of (90)Y-B3 and taxol to enhance the synergistic effect.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Fonoforese/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/terapia , Humanos , Marcação por Isótopo , Taxa de Depuração Metabólica/efeitos da radiação , Camundongos , Camundongos Nus , Especificidade de Órgãos/efeitos da radiação , Radioimunoterapia/métodos , Cintilografia , Distribuição Tecidual/efeitos da radiaçãoRESUMO
PURPOSE: Epidermal growth factor receptors (EGFR) play an important role in tumorigenesis and, therefore, have become targets for new molecular therapies. Here, we use a "cocktail" of optically labeled monoclonal antibodies directed against EGFR-1 (HER1) and EGFR-2 (HER2) to distinguish tumors by their cell surface expression profiles. EXPERIMENTAL DESIGN: In vivo imaging experiments were done in tumor-bearing mice following s.c. injection of A431 (overexpressing HER1), NIH3T3/HER2+ (overexpressing HER2), and Balb3T3/DsRed (non-expression control) cell lines. After tumor establishment, a cocktail of optically labeled antibodies: Cy5.5-labeled cetuximab (anti-HER1) and Cy7-labeled trastuzumab (anti-HER2) was i.v. injected. In vivo and ex vivo fluorescence imaging was done. For comparison with radionuclide imaging, experiments were undertaken using (111)Indium-labeled antibodies. Additionally, a "blinded" diagnostic study was done for mice bearing one tumor type. RESULTS: In vivo spectral fluorescent molecular imaging of 14 mice with three tumor types clearly differentiated tumors using the cocktail of optically labeled antibodies both in vivo and ex vivo. Twenty-four hours after injection, A431 and NIH3T3/HER2+ tumors were detected distinctly by their peak on Cy5.5 and Cy7 spectral images, respectively; radionuclide imaging was unable to clearly distinguish tumors at this time point. In blinded single tumor experiments, investigators were able to correctly diagnose a total of 40 tumors. CONCLUSION: An in vivo imaging technique using an antibody cocktail simultaneously differentiated two tumors expressing distinct EGFRs and enabled an accurate characterization of each subtype.
Assuntos
Anticorpos Monoclonais , Receptores ErbB/análise , Neoplasias/diagnóstico , Receptor ErbB-2/análise , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Carbocianinas/análise , Carbocianinas/química , Linhagem Celular Tumoral , Cetuximab , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos , Sondas Moleculares/análise , Sondas Moleculares/química , Neoplasias/diagnóstico por imagem , Cintilografia , TrastuzumabRESUMO
We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of 89Zr-B3 were dose-independent when 3 doses, 2, 15, and 60 µg B3, were compared at 24 h after injection. In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 µg) was needed to achieve tumor targeting comparable to the low dose (2 µg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 µg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 µg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.
Assuntos
Anticorpos Monoclonais/farmacocinética , Zircônio , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/imunologia , Proteínas Ligadas por GPI/imunologia , Meia-Vida , Xenoenxertos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Mesotelina , Mesotelioma/imunologia , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Radioisótopos , Distribuição TecidualRESUMO
[This corrects the article DOI: 10.18632/oncotarget.19945.].
RESUMO
OBJECTIVES: The aim of this research was to synthesize radiolabeled peptidomimetic integrin alpha(v)beta(3) antagonists that selectively target integrin alpha(v)beta(3) receptor and clear rapidly from the whole body. METHODS: Integrin alpha(v)beta(3) antagonists, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-beta-alanine (IA) and 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-beta-alanine hydrochloride (IAC), a hydrophobic carbamate derivative of IA, were conjugated with 2-p-isothiocyanatobenzyl-DOTA at the amino terminus and labeled with (111)In. The (111)In labeled IA and IAC were subjected to in vitro receptor binding, biodistribution and imaging studies using nude mice bearing the receptor-positive M21 human melanoma xenografts. RESULTS: The (111)In-labeled IA (40%) and -IAC (72%) specifically bound in vitro to alpha(v)beta(3) (0.8 microM) at a molar excess. This receptor binding was completely blocked by a molar excess of cold IA to alpha(v)beta(3). The higher receptor-binding affinity of the (111)In-labeled IAC was reflected in higher tumor uptake and retention: 5.6+/-1.4 and 4.5+/-0.7 %ID/g vs. 3.8+/-0.9 and 2.0+/-0.3 %ID/g for the (111)In-labeled IA at 0.33 and 2 h. The tumor uptakes were inhibited by the co-injection of 200 microg of IA, indicating that the uptake was receptor mediated. These antagonists were excreted primarily via the renal system. The (111)In activity retained in the whole body was quite comparable between the (111)In-labeled IA (24% ID) and the (111)In-labeled IAC (33% ID) at 2 h. The higher peak tumor uptake and longer retention resulted in higher tumor-to-background ratios for the (111)In-labeled IAC at 2 h with 9.7, 2.3, 0.8, 1.9, 7.1, 2.2, 0.9, 3.7 and 9.9 for blood, liver, kidney, lung, heart, stomach, intestine, bone and muscle, respectively. The imaging studies with the (111)In-labeled IAC also clearly visualized the receptor-positive tumor at 4 h. CONCLUSIONS: The (111)In-labeled IAC showed an improve tumor targeting kinetics with rapid accumulation and prolonged retention in the alpha(v)beta(3) receptor-positive tumor. This together with the rapid whole-body clearance pharmacokinetics warrants further studies on this IAC analog for molecular imaging of tumor-induced angiogenic vessels and various malignant human tumors expressing the receptor.
Assuntos
Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacocinética , Sulfonamidas/farmacocinética , beta-Alanina/análogos & derivados , Animais , Humanos , Radioisótopos de Índio , Injeções Intraperitoneais , Marcação por Isótopo , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Nus , Mimetismo Molecular , Transplante de Neoplasias , Neoplasias/metabolismo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , beta-Alanina/farmacocinéticaRESUMO
PURPOSE: To investigate the combined antitumor activity in mice of immunotoxin SS1P and Taxol. METHODS: Immunodeficient mice were implanted with A431/K5 tumors expressing mesothelin. Established tumors were treated i.v. with immunotoxin SS1P alone, i.p. with Taxol alone, or with the two agents together. SS1P was radiolabeled with (111)In and used to study the effect of Taxol on its uptake by A431/K5 tumors. RESULTS: Using doses at which either agent alone caused stabilization of tumor growth, the combination was synergistic causing long-lasting complete remissions in many animals. In contrast, synergy was not observed when the same cells were treated with these agents in vitro. Tumor uptake of (111)In-SS1P was not affected by treatment with Taxol. CONCLUSION: The combination of Taxol and SS1P exerts a synergistic antitumor effect in animals but not in cell culture. This effect is not secondary to increased tumor uptake of the immunotoxin. Synergy could be due to improved immunotoxin distribution within the tumor or could involve factors released by other cell types in the tumors.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Carcinoma/tratamento farmacológico , Paclitaxel/administração & dosagem , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Injeções Intraperitoneais , Injeções Subcutâneas , Mesotelina , Camundongos , Camundongos Nus , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3, which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64Cu-plerixafor, should be important investigative tools.
RESUMO
We investigated the biodistribution of (88)Y/(111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin and therapy with (90)Y-labeled DOTA-biotin in tumor-bearing mice after B3-streptavidin antibody conjugate (B3-SA) pretargeting. B3 antibody, recognizing Lewis(y) antigen, was conjugated to streptavidin (B3-SA). For pretargeting, 400 micro g of the B3-SA was injected i.v. into mice bearing A431 tumor xenografts. After tumor localization of B3-SA, 100 micro g of synthetic clearing agent was injected i.v. to clear the unbound B3-SA from the circulation. Four h later, 1 micro g of radiolabeled DOTA-biotin was injected i.v. Radioimmunotherapy was performed with doses of 9.25 to 37 MBq of (90)Y-labeled DOTA-biotin. As a result, radiolabeled DOTA-biotin cleared rapidly. All of the normal tissues had <2.6% of the injected dose per gram, whereas tumor uptake reached approximately 15% ID/g. The total tumor uptake of radioactivity remained similar for 96 h or longer. In the first study, the median survival of the control group was 8 days, whereas it increased to >163 days in the 37 MBq (90)Y group (P < 0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of (90)Y and B3-SA were cured, and remained healthy for >180 days after therapy, compared with control groups, with =29.2 days mean survival time (P < 0.001). Tumor pretargeting with B3-SA and radiolabeled DOTA-biotin has shown favorable, specific, and fast targeting that has resulted in good tumor responses and, thus, serves as a rationale for human studies with the B3-SA pretargeting approach.