RESUMEN
Conventional photodynamic therapy (PDT) for cancer is limited by the insufficient efficacy and specificity of photosensitizers. We herein describe a highly effective and selective tumor-targeted PDT using a near-infrared (NIR) photosensitizer, IRDye700DX, conjugated to a human monoclonal antibody (Ab) specific for carcinoembryonic antigen (CEA). The antitumor effects of this Ab-assisted PDT, called photoimmunotherapy (PIT), were investigated in vitro and in vivo. The Ab-IRDye conjugate induced potent cytotoxicity against CEA-positive tumor cells after NIR-irradiation, whereas CEA-negative cells were not affected at all, even in the presence of excess photoimmunoconjugate. We found an equivalent phototoxicity and a predominant plasma membrane localization of Ab-IRDye after both one and six hours of incubation. Either no or little caspase activation and membrane peroxidation were observed in PIT-treated cells and a panel of scavengers for reactive oxygen species showed only partial inhibition of the phototoxic effect. Strikingly, Ab-IRDye retained significant phototoxicity even under hypoxia. We established a xenograft model, which allowed us to sensitively investigate the therapeutic efficacy of PIT by non-invasive bioluminescence imaging. Luciferase-expressing MKN-45-luc human gastric carcinoma cells were subcutaneously implanted into both flanks of nude mice. NIR-irradiation was performed for only the tumor on one side. In vivo imaging and measurement of the tumor size revealed that a single PIT treatment, with intraperitoneal administration of Ab-IRDye and subsequent NIR-irradiation, caused rapid cell death and significant inhibition of tumor growth, but only on the irradiated side. Together, these data suggest that Ab-IRDye-mediated PIT has great potential as an anticancer therapeutics targeting CEA-positive tumors.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Inmunoterapia , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Western Blotting , Antígeno Carcinoembrionario/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Colorantes Fluorescentes/uso terapéutico , Humanos , Inmunoconjugados/administración & dosificación , Peroxidación de Lípido , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/inmunología , Neoplasias/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Clonación Molecular/métodos , Molécula de Adhesión Celular Epitelial , Humanos , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
BACKGROUND: Carcinoembryonic antigen (CEA) is frequently overexpressed in various types of human cancers and is associated with cell adhesion. There are three possible mechanisms of cancer therapy that employ anti-CEA antibody (Ab): Ab-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or the prevention of CEA interaction with the extracellular matrix and/or intercellular adhesion molecules resulting in anoikis. In this study, the effect of C2-74, a human anti-CEA monoclonal Ab was evaluated. METHODS: ADCC, CDC and anoikis assays in combination with C2-74 and an anticancer drug (5-fluorouracil or cisplatin) were investigated using tumor cell lines (MKN-45, MKN-74 and KATO III). In the anoikis assay, other human anti-CEA Abs and mouse anti-CEA-related cell adhesion molecule 6 Abs were also investigated using HLC-1 cells. RESULTS: Additive cytotoxicity was observed when the anticancer drug and C2-74 on tumor cells were combined in the CDC assays, whereas in the anoikis assay, no such additive effect was observed. Anti-CEA-related cell adhesion molecule 6 Abs, but not anti-CEA Abs, accelerated anoikis in HLC-1 cells. CONCLUSION: A mechanism for the additive antitumor effect when an anticancer drug and C2-74 are combined is indicated mainly by CDC activity but is irrelevant to anoikis in tumor cells.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Antígeno Carcinoembrionario/inmunología , Anoicis/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Cisplatino/farmacología , Complemento C2/inmunología , Fluorouracilo/farmacología , HumanosRESUMEN
BACKGROUND: The molecular and morphological alterations of the tight junctions in colorectal cancer (CRC) are still poorly understood. The possible involvement of claudin-1 (CL-1), one of the major tight junctional proteins (TJPs), was investigated in the tumorigenesis of CRC. PATIENTS AND METHODS: Adenocarcinoma tissue and paired normal mucosa specimens were resected from surgical specimens of CRC patients and analyzed to determine whether the expression of CL-1 correlated with the clinicopathological factors and to determine the role of CL-1 in the alteration of tight junctions during tumorigenesis. RESULTS: The expression of CL-1 at the mRNA and protein levels was analyzed in 41 cases and was found to increase in the CRC tissue in comparison to that in the normal tissue specimens. The mRNA levels of CL-1 were correlated with tumor depth, but not with the preoperative carcinoembryonic antigen (CEA) serum level. When T84 cells, a human colon cancer cell line, were transfected with the CL-1 gene, the CL-1 overexpressing cells grew as aggregates in contrast to the monolayer formation of the parental cells. In addition, trypsin-treated CL-1 overexpressing cells aggregated more easily than did the parental cells. CONCLUSION: CL-1 plays a pivotal role in cell morphology and behavior in the colonic epithelium. CL-1 protein may therefore be one of the major factors involved in the tumorigenesis of CRC.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas de la Membrana/metabolismo , Adenocarcinoma/secundario , Anciano , Anciano de 80 o más Años , Western Blotting , Claudina-1 , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/genética , Femenino , Humanos , Técnicas para Inmunoenzimas , Metástasis Linfática , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recto/metabolismo , Recto/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
IgA is able to trigger antibody-dependent cellular cytotoxicity (ADCC) by recruiting neutrophils expressing the Fc receptor for the CHalpha chain. We herein describe the preparation of a human recombinant anti-CEA IgA antibody to kill carcinoembryonic antigen (CEA)-expressing tumor cells via ADCC by neutrophils. A single chain Fv (scFv) gene was constructed using a cDNA library of a hybridoma clone that produces a human anti-CEA monoclonal IgG4 (C2-45). The scFv gene, linked with a CHalpha gene, was inserted into the pBK283 vector, which was cotransfected into BmN4 insect cells with the wild-virus BmNPV. After cloning and amplification, the recombinated virus was injected into silkworm larvae. The resulting human recombinant IgA, designated as 45scFvLCHalpha, was purified from hemolymph by Ni-affinity chromatography and characterized by ELISA, Western blotting, and the ADCC assay. 45scFvLCHalpha with an IgA antigenicity was bound to CEA and showed effective killing of the CEA-expressing cells in the presence of IFN-gamma-activated neutrophils. These data suggest the recombinant anti-CEA IgA antibody recruiting neutrophils maybe a useful means for the antibody-based immunotherapy of human CEA-expressing tumors.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno Carcinoembrionario/inmunología , Inmunoglobulina A/inmunología , Neutrófilos/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Bombyx , Células CHO , Antígeno Carcinoembrionario/biosíntesis , Antígeno Carcinoembrionario/genética , Línea Celular Tumoral , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina A/genética , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Inmunoterapia/métodos , Interferón gamma/farmacología , Neutrófilos/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Chemokines and their receptors are essential for leukocyte trafficking, and are also involved in cancer metastasis to specific organs. Although the migration of tumor cells into the lymph nodes is an important aspect of cancer, the processes involved are poorly understood. Chemokine receptors CCR7 and CXCR3 have been shown to play an important role in tumor cell migration and lymph node metastasis. Therefore, the assessment of chemokine receptor expression on lung adenocarcinomas may improve the prediction of the spread of this carcinoma to the lymph nodes. In this study, we examined the expression and function of these two chemokine receptors (CCR7 and CXCR3) in lung adenocarcinoma. By using flow cytometry, they were detected in all of the lung adenocarcinoma cell lines examined. In the chemotaxis assays, A549 cells exhibited CCL21-induced migration, which was significantly suppressed by neutralizing anti-CCR7 antibody. The CXCL10-induced migration of A549 cells was also significantly suppressed by neutralizing anti-CXCR3 antibody. In clinical lung adenocarcinoma samples, we found the expression of CCR7 and CXCR3 in 65 and 90% cases, respectively, most of which had lymph node metastasis. Importantly, the expression of CCR7 was significantly associated with lymph node metastasis, although the expression of CXCR3 was not. These results suggest that the activation of CCR7 and CXCR3 with their ligands preferentially stimulates lung adenocarcinoma metastasis to the draining lymph nodes.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Ganglios Linfáticos/metabolismo , Receptores CCR7/metabolismo , Receptores CXCR3/metabolismo , Adenocarcinoma/secundario , Quimiocina CCL21/metabolismo , Quimiotaxis , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/patología , Ganglios Linfáticos/patología , Metástasis Linfática , Pronóstico , Células Tumorales CultivadasRESUMEN
PURPOSE: Retinoic acid (RA) affects the activation of transforming growth factor-beta 1 (TGF-beta1) in a variety of cells. We have previously shown that thrombospondin-1 (TSP-1) expression in retinal pigment epithelial (RPE) cells is up-regulated by RA. Since TSP-1 activates TGF-beta1, we investigated whether RA stimulates the activation of TGF-beta1 through up-regulation of TSP-1 in RPE cells. METHODS: Human RPE cells were cultured with RA or TSP-1. The active form of TGF-beta1 in the culture medium was measured by enzyme-linked immunosorbent assay. RESULTS: The active form of TGF-beta1 was increased dose-dependently by RA or TSP-1. The activation of TGF-beta1 by RA was significantly hampered by an anti-TSP-1 antibody. Also, antibodies against integrins alphavbeta3 and alphavbeta5 inhibited the activation of TGF-beta1. CONCLUSIONS: These findings suggest that, in RPE cells, RA increases the activation of TGF-beta1 via up-regulation of TSP-1, and integrins such as alphavbeta3 and alphavbeta5 are essential in this activation step.
Asunto(s)
Epitelio Pigmentado Ocular/efectos de los fármacos , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Tretinoina/farmacología , Regulación hacia Arriba , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Integrina alfaVbeta3/fisiología , Integrinas/fisiología , Epitelio Pigmentado Ocular/metabolismo , Receptores de Vitronectina/fisiología , Trombospondina 1/farmacologíaRESUMEN
Gene therapy has the potential to provide highly selective, curative cancer treatments without inducing systemic toxicity. Adenoviral vectors have been extensively used for cancer gene therapy because of their relatively high efficacy of gene transfer. However, gene transduction to cancer cells is limited by the necessity of using adenoviral type 5 vectors. This is because these vectors have a low transduction efficiency due to weak expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR), on cancer cells. Moreover, there may be side-effects to the treatment as normal cells also express CAR. In order to eradicate cancer cells without side-effects, the development of a targeting-vector is therefore crucial. In this review, the recent targeting strategies of adenoviral vectors for cancer gene therapy are summarized.
Asunto(s)
Adenoviridae , Marcación de Gen , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Neoplasias/terapia , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Ligandos , Ratones , Modelos Biológicos , Receptores Virales , TropismoRESUMEN
Vascular endothelial growth factor (VEGF)-C and VEGF-D influence lymphangiogenesis through the activation of the vascular endothelial growth factor receptor (VEGFR)-3. They have been implicated in lymphatic tumor spread, which is an important prognostic factor in patients with non-small cell lung carcinoma (NSCLC). Whether or not the expression of VEGF-C, -D, and VEGFR-3 correlates with clinicopathological factors in patients with T1 lung adenocarcinoma was analysed. The tumor specimens were homogenized to determine the protein expression of VEGF-C, -D, and VEGFR-3 by enzyme-linked immunosorbent assay (ELISA). RNA fractions extracted from the tumor tissues were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) to assess the mRNA levels of VEGF-C, -D, and VEGFR-3. The expression of VEGF-D protein and mRNA levels in patients without lymph node metastasis were significantly higher than those with metastasis (p=0.013, p=0.0494, respectively). However, the protein and mRNA levels of VEGF-C and VEGFR-3 were not significantly different in patients with or without metastasis. The 5-year survival rates of the patients with high VEGF-D levels were significantly higher than those of patients with low levels (p =0.0221). No significant difference in the survival rates was observed for VEGF-C and VEGFR-3. VEGF-D may be downregulated in NSCLC tissues in comparison to adjacent normal tissue, resulting in lymph node metastasis and poor prognosis.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Metástasis Linfática/genética , Factor C de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Anciano , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
Sonodynamic therapy (SDT) of cancer is based on preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and subsequent activation of the drug by ultrasound irradiation. Ultrasound can penetrate deeply into tissues and can be focused into a small region of a tumor to activate a sonosensitizer. This is a unique advantage in the non-invasive treatment of nonsuperficial tumors when compared to laser light used for photodynamic therapy. Recently, it has been found that photochemically active porphyrins also show significant antitumor effects when activated with ultrasound. The mechanism of sonodynamic action has been suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. This mini-review provides a brief overview of the following four sonosensitizers useful in SDT: i) a homogeneous complex of oligomers of hematoporphyrin, Photofrin II; ii) a gallium porphyrin complex, ATX-70; iii) a hydrophilic chlorin derivative, A7X-S10, and iv) a novel porphyrin derivative devoid of photosensitivity, DCPH-P-Na (I).
Asunto(s)
Hematoporfirinas/uso terapéutico , Neoplasias/terapia , Fármacos Fotosensibilizantes/uso terapéutico , Terapia por Ultrasonido , Animales , Éter de Dihematoporfirina/química , Éter de Dihematoporfirina/uso terapéutico , Ratones , Modelos Biológicos , Porfirinas/química , Porfirinas/uso terapéutico , Ratas , UltrasonidoRESUMEN
BACKGROUND: To understand the molecular and morphological alterations in the tight junction in colorectal cancer (CRC) tissues, the expression of eight tight junction proteins in normal and cancer colorectal tissues were compared. PATIENTS AND METHODS: Adenocarcinoma tissues and paired normal mucosa were resected from surgical specimens of CRC patients. The expression of occludin, ZO-1, ZO-2, and claudin-1 -5 was analyzed at the mRNA level by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry. RESULTS: The expression of claudin-1 and claudin-2 in cancer tissues was upregulated 40- and 49.2-fold, respectively, at the mRNA level, as compared with that in normal tissues. The up-regulation of these two claudins was also observed at the protein level and it appeared to depend on the depth of tumor invasion. CONCLUSION: Claudin-1 and claudin-2 were found to be overexpressed in CRC tissues. They may be useful as tumor markers and targets for the treatment of colorectal cancer.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Proteínas de la Membrana/genética , Uniones Estrechas/metabolismo , Regulación hacia Arriba , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Claudina-1 , Claudinas , Femenino , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Ocludina , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de la Zonula Occludens-1RESUMEN
PURPOSE: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. EXPERIMENTAL DESIGN: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti-carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. RESULTS: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC(50) against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). CONCLUSIONS: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.
Asunto(s)
Antígeno Carcinoembrionario/genética , Terapia Genética/métodos , Neoplasias Gástricas/terapia , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular Tumoral , Cricetinae , Cartilla de ADN , Vectores Genéticos , Globinas/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy.
RESUMEN
BACKGROUND: Recombinant chimeric immune receptors (CIRs) with anti-CEA specificity can retarget grafted T-cells to CEA-expressing tumors in an HLA-independent manner. To reduce the immunogenicity of conventional CIR in humans, an attempt was made to generate a CIR encoded by all human genes. MATERIALS AND METHODS: A single-chain variable fragmented (scFv) antibody gene was prepared from variable region genes of the C2-45 human mAb clone specific for CEA. The scFv gene was connected to a gene construct comprised of the cDNAs for the human CD8a hinge region, the human CD28 transmembrane and cytoplasmic domains, and the human CD3zeta intracellular domain. The resulting human CIR gene, designated L45scFv-CIR, was inserted into the pcDNA3.1 expression vector and transfected into human primary T-cells. RESULTS: Flow cytometric analysis using allophycocyanin-labeled CEA demonstrated the expression of the L45scFv-CIR protein on the T-cells and its specific antigen binding activity. CONCLUSION: This L45scFv-CIR gene, consisting of four human genes, may be a useful tool for eradication of CEA-expressing but HLA-downregulated tumor cells.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Receptores Inmunológicos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/terapia , Linfocitos T/inmunología , Antígenos CD28/genética , Antígenos CD28/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Antígeno Carcinoembrionario/biosíntesis , Línea Celular Tumoral , Epítopos , Humanos , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/genética , Neoplasias Gástricas/genética , Linfocitos T/fisiología , TransfecciónRESUMEN
In this review article the possible applications of anti-tumor-associated antigen (TAA) antibodies in the therapy of cancer have been summarized. First, recombinant monoclonal antibodies (MAbs) are increasingly being used as therapeutic agents, especially in combination with anti-cancer drugs. Second, conjugation of antibody therapy with toxins or radioisotopes offers more therapeutic approaches. Third, development of cytotoxic T-lymphocyte (CTL) or natural killer (NK)-cell populations with anti-TAA antibody activity may be important for the success of cancer immunotherapy because the downregulated HLA class I molecules and the non-ubiquitous expression of NK receptor ligands in tumor tissues constitute the major tumor escape mechanism facing tumor-specific CTL- and/or NK-cell-mediated responses. Finally, in cancer gene therapy, the strategies to target viral vectors carrying therapeutic genes to tumor tissues by modifying the tropisms with MAbs or their genes against TAAs are also very promising.
Asunto(s)
Anticuerpos/genética , Anticuerpos/uso terapéutico , Antígenos de Neoplasias/inmunología , Inmunización Pasiva/métodos , Neoplasias/inmunología , Neoplasias/terapia , Animales , Anticuerpos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , HumanosRESUMEN
BACKGROUND: The IgG isotype of antibodies is very important for their biological functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). To increase the biological activity of a novel human monoclonal antibody (C2-45) against carcinoembryonic antigen (CEA), we tried to genetically convert its isotype from IgG4 to IgG1. MATERIALS AND METHODS: VH and VL genes were cloned from the parental antibody C2-45 (IgG4) and inserted into the pAc-kappa-CH3 expression vector which contained the constant region gene of human IgG1. The recombinant gene was transfected into Sf9 insect cells to produce recombinant protein. The resulting recombinant protein, designated C2-45 (cIgG1), in the culture medium was purified by affinity chromatography and characterized for its CEA binding activity and biological activity. RESULTS: The converted C2-45 (cIgG1) retained the original antigen-binding activity and showed significantly higher CDC and ADCC activities against CEA-expressing tumor cells than did the original C2-45 (IgG4). CONCLUSION: C2-45 (cIgG1) may be useful for antibody-based immunotherapy of human CEA-expressing tumors.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígeno Carcinoembrionario/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Células CHO , Línea Celular Tumoral , Clonación Molecular , Proteínas del Sistema Complemento/inmunología , Cricetinae , Citometría de Flujo , Humanos , Células Asesinas Activadas por Linfocinas/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , SpodopteraRESUMEN
OBJECTIVE: To analyze the prognostic value of hypoxia-inducible factor-1 (HIF-1) alpha expression and its correlation with clinicopathologic variables and the expression of vascular endothelial growth factor-A and -C in patients with lung adenocarcinomas of small size. METHODS: The expression of hypoxia-inducible factor-1 alpha was immunohistochemically determined in 78 cases of small-sized adenocarcinoma (maximum dimension < or = 2 cm) using antibody against a recombinant protein corresponding to amino acids 575-780 of hypoxia-inducible factor-1 alpha. Data regarding patient survival, clinicopathologic factors, and immunohistochemical studies of vascular endothelial growth factors were also collected. RESULTS: Strong expression of hypoxia-inducible factor-1 alpha was observed in 23 of 78 cases; absent or minimal expression was found in the localized bronchioloalveolar carcinomas. Strong expression of hypoxia-inducible factor-1 alpha was significantly higher in cases with vascular invasion, lymph node involvement, and vascular endothelial growth factor-A expression. The 5-year survival rate was 63.2% if expression of hypoxia-inducible factor-1 alpha was strong and 85.1% if expression was weak (p < 0.05). CONCLUSION: Immunohistochemical staining of HIF-1 alpha, along with examination of metastatic potential via vascular pathways, may be valid defining a subpopulation of patients with small-sized adenocarcinoma of the lung whose tumors have aggressive angiogenesis potential.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/secundario , Adenocarcinoma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Metástasis Linfática , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Pronóstico , Análisis de Supervivencia , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cellular immunity, in which cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are main effector cells, plays an important role in the antitumor defense mechanism. T cell immunotherapy is based on the assumption that tumor antigen (TA) peptides are correctly presented by HLA class I molecules on target tumor cells, while NK cell immunotherapy is based on the hypothesis that cell surface TAs or ligands for NK receptors are widely expressed in tumor cells. However, human tumor cells are well known to often lose HLA class 1 molecules, and target cell ligands for NK receptors are not always expressed in human tumor cells. This altered HLA class 1 expression and non-ubiquitous distribution of NK receptor ligands constitute the major tumor escape mechanism facing tumor-specific CTL and/or NK cell-mediated responses. These facts also indicate that it is not easy to eliminate the target tumors only by activating tumor-specific CTLs or NK cells. On the other hand, although the protective role of humoral immunity in cancer seems not to be imperative, it is easily confirmed by immunostaining whether or not antibody-recognized TAs such as carcinoembryonic antigen (CEA) exist on the cell surface of target tumor cells. Therefore, endowing CTLs or NK cells with antigen-binding specificity of anti-TA antibody is promising for re-targeting the activities of these effector cells to tumor cells in an HLA-independent manner. This mini-review provides a brief overview of the following four technologies for re-targeting CTLs or NK cells to CEA-expressing tumor cells with anti-CEA antibody activity: i) bispecific antibody technology, ii) antibody-cytokine fusion protein technology, iii) chimeric immune receptor technology, and iv) antibody-HLA/peptide complex technology.
Asunto(s)
Anticuerpos/inmunología , Antígeno Carcinoembrionario/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígeno Carcinoembrionario/biosíntesis , Antígenos HLA/inmunología , Humanos , Inmunoconjugados/inmunología , Inmunoconjugados/uso terapéutico , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/uso terapéuticoRESUMEN
For antibody-based therapy of cancer, monoclonal antibodies (mAbs) of human origin are superior to mouse, mouse/human chimeric or humanized mAbs, because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study, human mAbs were prepared against a pancarcinoma antigen, MK-1 (Ep-CAM), using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice, immunized with recombinant MK-1, were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed, two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants, their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, respectively, against MK-1-expressing cells in vitro, suggesting that these fully human mAbs produced against MK-1 and their V-region genes, which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Células CHO , Línea Celular Tumoral , Clonación Molecular , Cricetinae , Molécula de Adhesión Celular Epitelial , Técnicas de Transferencia de Gen , Humanos , Inmunoglobulina G/genética , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/terapiaRESUMEN
[This corrects the article DOI: 10.1155/2012/853879.].