RESUMO
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Assuntos
Complexo CD3 , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/terapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Complexo CD3/imunologia , Animais , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Ativação Linfocitária/imunologia , Feminino , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Neoplasias , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/farmacologia , Capecitabina , Humanos , Neoplasias/tratamento farmacológico , Linfócitos TRESUMO
Methyl mercury (MeHg) poisoning or Minamata disease (MD) from fish consumption is a public health concern throughout the world because all fish contain small amounts. The lowest exposure level needed to impair children's development is controversial. Actual poisoning with MeHg from fish consumption has been reliably reported only two times. It occurred in Minamata, Japan in the 1950s and then in Niigata, Japan in the 1960s. On each occasion, massive industrial pollution led to local fish having mercury levels as high as 40ppm. In Niigata the pollution was on the Agano River and there were over 2000 commercial fishermen active at that time. We studied adult subjects who had been exposed perinatally to MeHg from fish consumption during the Niigata poisoning to determine the long-term impact of exposure. We identified mothers with elevated levels of exposure during the epidemic and those diagnosed with MeHg poisoning. The subjects of the study were their adult children, born during the epidemic. The evaluation consisted of a questionnaire (administered by interview) focusing on development, symptoms, and current function and a standard medical and neurological examination. The subjects were divided into four groups based upon prenatal levels of mercury in maternal hair or the presence of MD. For Group A the hair mercury levels were 50ppm or more, for Group B the mercury levels were 25-49ppm, and for Group C 10-24ppm. The subjects in Group D were born to mothers diagnosed with MD, but their mercury levels were not measured. Exposure was predominantly prenatal, but some mothers also breast fed their infants. Group A included 13 subjects among whom two were diagnosed with congenital MeHg poisoning and in two others it was suspected. Group B included 10 subjects, of whom three had symptoms compatible with MeHg poisoning. Group C had nine subjects including one with intellectual deficit and another with hearing loss. Group D had eight subjects of whom four had symptoms compatible with MeHg exposure, but only one had abnormal neurological findings. Among the subjects thought to have congenital or childhood MeHg poisoning, intelligence did not appear to have declined over time. More children were affected by prenatal and postnatal MeHg exposure at Niigata than was previously reported.
Assuntos
Intoxicação do Sistema Nervoso por Mercúrio , Mercúrio , Compostos de Metilmercúrio , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Humanos , Gravidez , Peixes , Contaminação de Alimentos , Cabelo/química , Japão/epidemiologia , Mercúrio/análise , Intoxicação do Sistema Nervoso por Mercúrio/diagnóstico , Intoxicação do Sistema Nervoso por Mercúrio/epidemiologia , Compostos de Metilmercúrio/análise , Compostos de Metilmercúrio/toxicidadeRESUMO
This corrects the article DOI: 10.1038/srep45839.
RESUMO
We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1⯵g/g body weight) or a high dose (1⯵g/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6â¯M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.
Assuntos
Reabsorção Óssea/patologia , Carpa Dourada/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , alfa-MSH/farmacologia , Fosfatase Alcalina/metabolismo , Escamas de Animais/metabolismo , Animais , Reabsorção Óssea/genética , Cálcio/sangue , Cálcio/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Carpa Dourada/sangue , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração/efeitos dos fármacosRESUMO
Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Glipicanas/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Complexo CD3/metabolismo , Citocinas/metabolismo , Humanos , Imunocompetência/efeitos dos fármacos , Injeções Intravenosas , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Macaca fascicularis , Camundongos Transgênicos , Esteroides/farmacologia , Esteroides/uso terapêutico , Linfócitos T/efeitos dos fármacosRESUMO
T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.
Assuntos
Complexo CD3/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , CamundongosRESUMO
CD47 belongs to the immunoglobulin superfamily and is associated with ß-integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B-chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47-mediated apoptosis in human lymphoid malignant cells. Treatment with S-S diabody alone induced apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of S-S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK-1 cells. In gene expression profiling of the S-S diabody-treated MOLT-4 cells, hypoxia inducible factor (HIF)-1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF-1α, RTP801 and BNIP3 were increased. Knockdown of HIF-1α by siRNA repressed S-S diabody-induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S-S diabody might have potential as a novel therapeutic agent for B-CLL.
Assuntos
Apoptose , Antígeno CD47/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia Linfocítica Crônica de Células B/terapia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Potenciais da Membrana , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Microscopia Eletrônica , Proteínas Mitocondriais/genética , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Fatores de Transcrição/genéticaRESUMO
Glypican 3 (GPC3), a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, is expressed in a majority of hepatocellular carcinoma tissues. The murine monoclonal antibody GC33 that specifically binds to the COOH-terminal part of GPC3 causes strong antibody-dependent cellular cytotoxicity against hepatocellular carcinoma cells and exhibits strong antitumor activity in the xenograft models. To apply GC33 for clinical use, we generated a humanized GC33 from complementarity-determining region grafting with the aid of both the hybrid variable region and two-step design methods. The humanized antibody bound to GPC3 specifically and induced antibody-dependent cellular cytotoxicity as effectively as a chimeric GC33 antibody. To improve stability of the humanized GC33, we further optimized humanized GC33 by replacing the amino acid residues that may affect the structure of the variable region of a heavy chain. Substitution of Glu6 with Gln in the heavy chain significantly improved the stability under high temperatures. GC33 also has the risk of deamidation of the -Asn-Gly- sequence in the complementarity-determining region 1 of the light chain. As substitution of Asn diminished the antigen binding, we changed the neighboring Gly to Arg to avoid deamidation. The resulting humanized anti-GPC3 antibody was as efficacious as chimeric GC33 against the HepG2 xenograft and is now being evaluated in clinical trials.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Glipicanas/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma Hepatocelular/patologia , Regiões Determinantes de Complementaridade/imunologia , Desenho de Fármacos , Humanos , Região Variável de Imunoglobulina/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Estabilidade Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC) and data on the expression profile in HCC might be useful for therapeutic decision-making and prognostic prediction. This study was performed using HepG2 xenograft tissues to optimize the tissue processing method for GPC3 immunohistochemistry. The optimization was conducted in terms of using GPC3 immunohistochemistry for biological study of GPC3 (Experiment 1) and as a diagnostic tool (Experiment 2). In Experiment 1, GPC3 immunoreactivity (IR) and tissue architecture were compared among differently fixed and embedded specimens. In Experiment 2, using conventional formalin-fixed paraffin-embedded (FFPE) procedures, the effects of different fixation times and antigen retrieval treatments were assessed. In Experiment 1, the periodate-lysine-paraformaldehyde (PLP)-fixed and AMeX method-embedded (PLP-AMeX) specimen showed superior immunoreactivity and excellent tissue architecture preservation. In contrast, the other specimens, especially frozen specimens, resulted in poor IR. In Experiment 2, specimens fixed for 24h showed better IR than those fixed for 7 days and the most remarkable improvement in IR was achieved after protease treatment. These findings indicate that with GPC3 immunohistochemistry for biological studies, the PLP-AMeX specimen is preferable. For diagnostics using FFPE specimens, the fixation time should not be too long and protease should be used for the antigen retrieval treatment.
Assuntos
Glipicanas/análise , Imuno-Histoquímica/métodos , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Animais , Anticorpos Monoclonais , Fixadores , Formaldeído , Glipicanas/imunologia , Glipicanas/metabolismo , Células Hep G2 , Humanos , Lisina , Masculino , Camundongos , Camundongos SCID , Ácido Periódico , Fatores de Tempo , TransplantesRESUMO
Previously, we demonstrated the antitumor efficacy of the anti-glypican-3 (GPC3) antibody GC33 in several human liver cancer xenograft models and the important role of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor mechanism of GC33. Involvement of other mechanisms such as modulation of the functions of GPC3 in antitumor activity remains to be elucidated. In this study, we investigated histopathologically time-course changes in xenografts in mice following a single administration of GC33 to clarify the morphological changes contributing to the tumor growth inhibition of GC33, including the changes in GPC3-related factors/components [proliferation, extracellular matrices (ECMs) and macrophage]. Histopathological changes peaked 3-5 d after GC33 administration and included increased tumor cell death, tumor cells with round morphology, multinucleated tumor cells and small spindle/round-like cells (mostly F4/80-positive macrophages). No direct effects of GC33 on proliferation activity of tumor cells were observed. Meanwhile, alteration of ECM structures and a remarkable increase in macrophages was noted in the GC33-treated group. Increase in macrophages was observed mainly in the outer layer of tumor nodules; the area of the increase approximately included the area where the change in tumor cells and ECMs were observed. Interestingly, depletion of macrophages in the xenograft models resulted in a marked reduction of the antitumor activity of GC33. In the in vitro ADCC assay, ADCC was only slightly induced by mouse peritoneal macrophages. These data suggest that macrophages play an important role in the antitumor activity of GC33, which is not likely to be direct ADCC by macrophages themselves.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma Hepatocelular/patologia , Glipicanas/imunologia , Neoplasias Hepáticas/patologia , Macrófagos/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Glipicanas/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Neoplasias Hepáticas/imunologia , Camundongos , Proteínas de Neoplasias/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human glypican 3 (GPC3) is preferentially expressed in the tumor tissues of liver cancer patients. In this study, we obtained a monoclonal antibody (mAb) against the COOH-terminal part of GPC3, which induced antibody-dependent cellular cytotoxicity (ADCC). The mAb, designated GC33, exhibited marked tumor growth inhibition of s.c. transplanted Hep G2 and HuH-7 xenografts that expressed GPC3 but did not inhibit growth of the SK-HEP-1 that was negative for GPC3. GC33 was efficacious even in an orthotopic model; it markedly reduced the blood alpha-fetoprotein levels of mice intrahepatically transplanted with Hep G2 cells. Humanized GC33 (hGC33) was as efficacious as GC33 against the Hep G2 xenograft, but hGC33 lacking carbohydrate moieties caused neither ADCC nor tumor growth inhibition. Depletion of CD56+ cells from human peripheral blood mononuclear cells markedly abrogated the ADCC caused by hGC33. The results show that the antitumor activity of hGC33 is mainly attributable to ADCC, and in human, natural killer cell-mediated ADCC is one possible mechanism of the antitumor effects by GC33. hGC33 will provide a novel treatment option for liver cancer patients with GPC3-positive tumors.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Glipicanas/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Antígenos de Neoplasias/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzenossulfonatos/administração & dosagem , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Doxorrubicina/administração & dosagem , Glipicanas/biossíntese , Glipicanas/genética , Humanos , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos SCID , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piridinas/administração & dosagem , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ligation of CD47 induces the apoptosis of leukemic cells in a caspase-independent manner. We generated a monoclonal antibody against CD47 (mAb-MABL) that possibly induced apoptosis from the ligation of CD47 in CCRF-CEM and JOK-1 cells in vitro. To confirm whether the ligation of CD47 caused cell death in vivo, we examined the antitumor activity of F(ab')2 of mAb-MABL in two xenograft models: The acute lymphoblastic leukemia (CCRF-CEM) and the B-cell chronic lymphocytic leukemia (JOK-1) cell line. Furthermore, in order to clarify the apoptotic activity selective for the tumor cells, we examined F(ab')2 of mAb-MABL apoptotic effects on CD34+ hematopoietic progenitor/stem and human endothelial cells. Male SCID mice were intravenously injected with CCRF-CEM (5 x 10(6) cells/mouse) or JOK-1 cells (5 x 10(6) cells/mouse) and intraperitoneally with JOK-1 cells (2 x 10(7) cells/mice). After the implantation of the cells, the mice were intravenously administered the vehicle or the F(ab')2 fragment of mAb-MABL at several doses and the length of survival was measured. F(ab')2 of mAb-MABL markedly prolonged the survival of mice transplanted with CCRF-CEM and JOK-1. Significantly, 40% of the mice intraperitoneally injected with JOK-1 cells became tumor-free when administered F(ab')2 of mAb-MABL, whereas even a high dose of fludarabine only slightly prolonged the median survival time. On the contrary, F(ab')2 of mAb-MABL showed no apoptotic effect on CD34+ hematopoietic progenitor/stem or human endothelial cells. Thus, monoclonal antibodies that cause cell death from the ligation of CD47 could be novel therapeutic agents for incurable leukemia after further optimization such as humanization or making single chain diabodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Animais , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Leucemia L1210 , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma.
Assuntos
Antígenos HLA-A/imunologia , Imunização Passiva/métodos , Fragmentos de Imunoglobulinas/farmacologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Morte Celular/imunologia , Linhagem Celular Tumoral , Citocinas/farmacologia , Ativação Enzimática , Antígenos HLA-A/biossíntese , Humanos , Células Jurkat , Camundongos , Camundongos SCID , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Hepatocellular carcinoma is the most common primary malignancy of the liver and accounts for as many as one million deaths annually worldwide. The present study was done to identify new transmembrane molecules for antibody therapy in hepatocellular carcinoma. EXPERIMENTAL DESIGN: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules. RESULTS: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11). CONCLUSIONS: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Animais , Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Células COS , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Testes Imunológicos de Citotoxicidade , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/sangue , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Receptores Imunológicos/biossíntese , Receptores Imunológicos/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima/genética , Proteínas RoundaboutRESUMO
A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos/imunologia , Glicoproteínas de Membrana/imunologia , Mieloma Múltiplo/tratamento farmacológico , Animais , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD , Mapeamento de Epitopos , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma is currently considered incurable despite the use of high-dose chemotherapy with autologous hematopoietic stem cell transplantation support. Here, we show antitumor efficacy of a novel bivalent single-chain antibody fragment (scFv) against CD47 in an in vivo myeloma model. We generated two types of novel scFv molecules against CD47 having apoptosis-inducing activity for leukemic cell lines: a non-covalently linked scFv dimer (diabody) and a covalently linked bivalent scFv. Administration of these bivalent scFvs significantly prolonged the survival of mice transplanted with KPMM2 human myeloma cells. Because bivalent scFvs induced neither ADCC nor CDC, such antitumor activity by bivalent scFv is presumably attributable to cell death caused by the ligation of CD47. Thus, these apoptosis-inducing scFvs will be effective as a novel therapy for multiple myeloma which is considered incurable with conventional therapy.
Assuntos
Antígenos CD/imunologia , Apoptose/efeitos dos fármacos , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/uso terapêutico , Mieloma Múltiplo/patologia , Animais , Antineoplásicos/toxicidade , Antígeno CD47 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Leucemia L1210/tratamento farmacológico , Leucemia L1210/patologia , Masculino , Camundongos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Transplante HeterólogoRESUMO
A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.