RESUMO
Elotuzumab is a humanized monoclonal antibody specific for signaling lymphocytic activation molecule-F7 (SLAMF7, also known as CS1, CD319, or CRACC) that enhances natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) of SLAMF7-expressing myeloma cells. This study explored the mechanisms underlying enhanced myeloma cell killing with elotuzumab as a single agent and in combination with lenalidomide, to support ongoing phase III trials in patients with relapsed/refractory or newly-diagnosed multiple myeloma (MM). An in vitro peripheral blood lymphocyte (PBL)/myeloma cell co-culture model was developed to evaluate the combination of elotuzumab and lenalidomide. Expression of activation markers and adhesion receptors was evaluated by flow cytometry, cytokine expression by Luminex and ELISPOT assays, and cytotoxicity by myeloma cell counts. Elotuzumab activated NK cells and promoted myeloma cell death in PBL/myeloma cell co-cultures. The combination of elotuzumab plus lenalidomide demonstrated superior anti-myeloma activity on established MM xenografts in vivo and in PBL/myeloma cell co-cultures in vitro than either agent alone. The combination enhanced myeloma cell killing by modulating NK cell function that coincided with the upregulation of adhesion and activation markers, including interleukin (IL)-2Rα expression, IL-2 production by CD3(+)CD56(+) lymphocytes, and tumor necrosis factor (TNF)-α production. In co-culture assays, TNF-α directly increased NK cell activation and myeloma cell death with elotuzumab or elotuzumab plus lenalidomide, and neutralizing TNF-α decreased NK cell activation and myeloma cell death with elotuzumab. These results demonstrate that elotuzumab activates NK cells and induces myeloma cell death via NK cell-mediated ADCC, which is further enhanced when combined with lenalidomide.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Camundongos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/imunologia , Receptores Imunológicos/metabolismo , Western Blotting , Proliferação de Células , Citometria de Fluxo , Humanos , Imunoprecipitação , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Imunológicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Receptores Imunológicos/imunologia , Animais , Antígenos de Neoplasias , Medula Óssea , Humanos , Camundongos , Mieloma Múltiplo/patologia , RNA Mensageiro/análise , Receptores Imunológicos/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Células Estromais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models. EXPERIMENTAL DESIGN: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice. RESULTS: CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like T cells, and CD8+ T cells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice. CONCLUSIONS: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1 with HuLuc63 for the treatment of multiple myeloma.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Subpopulações de Linfócitos/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Plasmócitos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/citologia , Camundongos , Camundongos SCID , Mieloma Múltiplo/imunologia , Plasmócitos/citologia , Receptores Imunológicos/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Loss of the DNA-dependent protein kinase (DNA-PK) results in increased sensitivity to ionizing radiation due to inefficient repair of DNA double-strand breaks. Overexpression of DNA-PK in tumor cells conversely results in resistance to ionizing radiation. It is therefore possible that inhibition of DNA-PK will enhance the preferential killing of tumor cells by radiotherapy. Available inhibitors of DNA-PK, like wortmannin, are cytotoxic and stop the cell cycle because they inhibit phoshatidylinositol-3-kinases at 100-fold lower concentrations required to inhibit DNA-PK. In an effort to develop a specific DNA-PK inhibitor, we have characterized SU11752, from a three-substituted indolin-2-ones library. SU11752 and wortmannin were equally potent inhibitors of DNA-PK. In contrast, inhibition of the phoshatidylinositol-3-kinase p110gamma required 500-fold higher concentration of SU11752. Thus, SU11752 was a more selective inhibitor of DNA-PK than wortmannin. Inhibition kinetics and a direct assay for ATP binding showed that SU11752 inhibited DNA-PK by competing with ATP. SU11752 inhibited DNA double-strand break repair in cells and gave rise to a five-fold sensitization to ionizing radiation. At concentrations of SU11752 that inhibited DNA repair, cell cycle progression was still normal and ATM kinase activity was not inhibited. We conclude that SU11752 defines a new class of drugs that may serve as a starting point for the development of specific DNA-PK inhibitors.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA , Radiação IonizanteRESUMO
The hepatocyte growth factor/scatter factor (HGF/SF) receptor, Met, mediates various cellular responses on activation with its ligand, including proliferation, survival, motility, invasion, and tubular morphogenesis. Met expression is frequently up-regulated in sarcomas and carcinomas. Experimental evidence suggests that Met activation correlates with poor clinical outcome and the likelihood of metastasis. Therefore, inhibitors of Met tyrosine kinase may be useful for the treatment of a wide variety of cancers that have spread from the primary site. We have discovered potent and selective pyrrole-indolinone Met kinase inhibitors and characterized them for their ability to inhibit HGF/SF-induced cellular responses in vitro. These compounds inhibit HGF/SF-induced receptor phosphorylation in a dose-dependent manner. They also inhibit the HGF/SF-induced motility and invasion of epithelial and carcinoma cells. Therefore, these compounds represent a class of prototype small molecules that selectively inhibit the Met kinase and could lead to identification of compounds with potential therapeutic utility in treatment of cancers.
Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Invasividade Neoplásica , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas c-met/química , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Monoclonal antibody (mAb) therapy for multiple myeloma, a malignancy of plasma cells, has not been clinically efficacious in part due to a lack of appropriate targets. We recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, is currently in a phase I clinical trial in relapsed/refractory myeloma. In this report we investigated whether the activity of elotuzumab could be enhanced by bortezomib, a reversible proteasome inhibitor with significant activity in myeloma. We first showed that elotuzumab could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model. We next showed that CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity, both for OPM2 myeloma cells using natural killer or peripheral blood mononuclear cells from healthy donors and for primary myeloma cells using autologous natural killer effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo antitumor activity. These findings provide the rationale for a clinical trial combining elotuzumab and bortezomib, which will test the hypothesis that combining both drugs would result in enhanced immune lysis of myeloma by elotuzumab and direct targeting of myeloma by bortezomib.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácidos Borônicos/uso terapêutico , Glicoproteínas de Membrana/imunologia , Mieloma Múltiplo/tratamento farmacológico , Pirazinas/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Apoptose/efeitos dos fármacos , Bortezomib , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos SCID , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Transplante HeterólogoRESUMO
A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.
Assuntos
Focalização Isoelétrica/métodos , Proteínas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Ponto Isoelétrico , Fosforilação , Isoformas de Proteínas/metabolismo , Sensibilidade e EspecificidadeRESUMO
Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Cristalino/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cristalino/fisiologia , Notophthalmus viridescens , Regeneração/fisiologiaRESUMO
SU9516 is a 3-substituted indolinone compound with demonstrated potent and selective inhibition toward cyclin dependent kinases (cdks). Here, we describe the kinetic characterization of this inhibition with respect to cdk2, 1, and 4, along with the crystal structure in complex with cdk2. The molecule is competitive with respect to ATP for cdk2/cyclin A, with a K(i) value of 0.031 microM. Similarly, SU9516 inhibits cdk2/cyclin E and cdk1/cyclin B1 in an ATP-competitive manner, although at a 2- to 8-fold reduced potency. In contrast, the compound exhibited non-competitive inhibition with respect to ATP toward cdk4/cyclin D1, with a 45-fold reduced potency. The X-ray crystal structure of SU9516 bound to cdk2 revealed interactions between the molecule and Leu83 and Glu81 of the kinase. This study should aid in the development of more potent and selective cdk inhibitors for potential therapeutic agents.
Assuntos
Quinases relacionadas a CDC2 e CDC28/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Imidazóis/farmacologia , Indóis/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Humanos , Insetos , Cinética , Modelos Químicos , Ligação ProteicaRESUMO
Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.
Assuntos
Movimento Celular/fisiologia , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Quinonas/química , Quinonas/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Cinética , Camundongos , Células NIH 3T3 , Oxirredução , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a ReceptoresRESUMO
Vascular endothelial growth factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity in diabetic retinopathy and neovascular age-related macular degeneration. Our aim was to identify an orally active, selective small molecule kinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-2 with activity against both VEGF-induced angiogenesis and vascular permeability. We used a biochemical assay to identify 3-[5-methyl-2- (2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-proprionic acid (SU10944), a pyrrole indolinone, which is a potent ATP-competitive inhibitor of VEGFR-2 (Ki of 21 +/- 5 nM). In cellular assays, SU10944 inhibited VEGF-induced receptor autophosphorylation (IC50 of 227 +/- 80 nM) as well as downstream signaling (IC50 of 102 +/- 27 nM). In biochemical assays, SU10944 exhibits potent inhibitory activity against VEGFR-1; weak activity against other related subgroup members, including stem cell factor receptor (SCFR), platelet-derived growth factor receptor beta (PDGFRbeta), and fibroblast growth factor receptor-1 (FGFR-1); and no detectable activity against other protein tyrosine kinases such as epidermal growth factor receptor (EGFR), Src, and hepatocyte growth factor receptor. In cellular assays, the selectivity for SU10944 to inhibit VEGFR is maintained compared with other tyrosine kinases (IC50 for SCFR of 1.6 +/- 0.3 microM, for PDGFRbeta of 30.6 +/- 13.3 microM, for FGFR-1 of >50 microM, and for EGFR of >50 microM). Upon oral administration, SU10944 gave a clear dose response in the corneal micropocket model with an ED50 value for inhibition of neovascularization of approximately 30 mg/kg and a maximum inhibition of 95% at 300 mg/kg. Similarly, upon oral administration in the Miles assay, SU10944 potently inhibited VEGF-induced vascular permeability. Our data indicate that small molecule inhibitors of VEGFR signaling have the potential to ameliorate VEGF-induced neovascularization as well as vascular permeability.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Indóis/farmacologia , Neovascularização Patológica/prevenção & controle , Propionatos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Células 3T3 , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Indóis/uso terapêutico , Camundongos , Propionatos/uso terapêuticoRESUMO
It was recently shown that vascular endothelial growth factor (VEGF), a growth factor for endothelial cells, plays a pivotal role in rheumatoid arthritis. VEGF binds to specific receptors, known as VEGF-RI and VEGF-RII. We assessed the physical and histological effects of selective blockade of VEGF and its receptors in transgenic K/BxN mice, a model of rheumatoid arthritis very close to the human disease. Mice were treated with anti-mouse VEGF Ab, anti-mouse VEGF-RI and -RII Abs, and an inhibitor of VEGF-RI tyrosine kinase. Disease activity was monitored using clinical indexes and by histological examination. We found that synovial cells from arthritic joints express VEGF, VEGF-RI, and VEGF-RII. Treatment with anti-VEGF-RI strongly attenuated the disease throughout the study period, while anti-VEGF only transiently delayed disease onset. Treatment with anti-VEGF-RII had no effect. Anti-VEGF-RI reduced the intensity of clinical manifestations and, based on qualitative and semiquantitative histological analyses, prevented joint damage. Treatment with a VEGF-RI tyrosine kinase inhibitor almost abolished the disease. These results show that VEGF is a key factor in pannus development, acting through the VEGF-RI pathway. The observation that in vivo administration of specific inhibitors targeting the VEGF-RI pathway suppressed arthritis and prevented bone destruction opens up new possibilities for the treatment of rheumatoid arthritis.