RESUMO
AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Epitélio Corneano/efeitos dos fármacos , Imunoconjugados/toxicidade , Pinocitose/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Macaca fascicularis , Masculino , Modelos Animais , Coelhos , Homologia de Sequência de AminoácidosRESUMO
Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877-86. ©2017 AACRSee related article by Zhao et al., p. 1866.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Imunoconjugados/farmacologia , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Pinocitose , Antineoplásicos Imunológicos/efeitos adversos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoconjugados/efeitos adversos , Megacariócitos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Transporte Proteico , Receptores de IgG/metabolismo , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamenteRESUMO
Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866-76. ©2017 AACRSee related article by Zhao et al., p. 1877.
Assuntos
Antineoplásicos/efeitos adversos , Imunoconjugados/efeitos adversos , Mielopoese/efeitos dos fármacos , Neutropenia/sangue , Neutropenia/etiologia , Neutrófilos/efeitos dos fármacos , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Camundongos , Neutrófilos/metabolismo , Pinocitose , Receptores de IgG/metabolismo , Serina Proteases/metabolismoRESUMO
Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Carcinoma/metabolismo , Imunoconjugados/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/farmacologia , Neoplasias Pancreáticas/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models. PROCEDURES: The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated. RESULTS: A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues. CONCLUSIONS: [(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.
Assuntos
Anticorpos Monoclonais/imunologia , Moléculas de Adesão Celular/imunologia , Tomografia por Emissão de Pósitrons/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Indicadores e Reagentes , Neoplasias Hepáticas/secundário , Macaca fascicularis , Camundongos , Nectinas , Distribuição Tecidual , Zircônio/químicaRESUMO
The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/imunologia , Animais , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Macaca fascicularis , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Nectinas , Neoplasias/enzimologia , Neoplasias/patologia , Ratos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Oligopeptídeos/química , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Neoplasias Renais/metabolismo , Camundongos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Regulação para Cima , Urotélio/metabolismo , Urotélio/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Imunoconjugados/farmacologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Pirofosfatases/antagonistas & inibidores , Animais , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/tratamento farmacológico , Macaca fascicularis , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Imunoconjugados/farmacologia , Leucemia Mieloide/tratamento farmacológico , Linfoma não Hodgkin/tratamento farmacológico , Tetraspaninas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Doença Aguda , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/imunologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/metabolismo , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/imunologia , Linfoma de Células T/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Tetraspaninas/metabolismo , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFß-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.
Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Receptores com Domínio Discoidina , Humanos , Camundongos SCID , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Transdução de SinaisRESUMO
Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)-hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated beta-catenin level while down-regulating nuclear factor-kappaB (NF-kappaB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Imunoterapia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Mieloma Múltiplo/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Diferenciação Celular , Células Cultivadas , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/imunologia , Osteogênese/efeitos dos fármacos , Osteogênese/imunologiaRESUMO
Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti-Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti-Dkk1 treatment prevented 5T2MM-induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. muCT analysis showed that anti-Dkk1 treatment significantly protected against 5T2MM-induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mieloma Múltiplo/complicações , Mieloma Múltiplo/fisiopatologia , Osteogênese , Osteólise/complicações , Osteólise/prevenção & controle , Animais , Anticorpos/farmacologia , Contagem de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mieloma Múltiplo/patologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteólise/patologia , Osteólise/fisiopatologia , Solubilidade/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Proteínas Wnt/metabolismoRESUMO
Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, which is being developed as an anti-tumour agent due to its selective toxicity to tumour cells, induces apoptosis by binding to two membrane-bound receptors, TRAIL-R1 and TRAIL-R2. Clinical trials have been initiated with various preparations of TRAIL as well as agonistic monoclonal antibodies to TRAIL-R1 and TRAIL-R2. Previously we reported that prior treatment of primary chronic lymphocytic leukaemia (CLL) cells with histone deacetylase inhibitors was required to sensitize CLL cells to TRAIL and, using various receptor-selective TRAIL mutant ligands, we demonstrated that CLL cells signalled to apoptosis primarily through TRAIL-R1. Some, but not all, agonistic TRAIL-receptor antibodies require cross-linking in order to induce apoptosis. The present study demonstrated that CLL cells can signal to apoptosis through the TRAIL-R2 receptor, but only after cross-linking of the agonistic TRAIL-R2 antibodies, LBY135 and lexatumumab (HGS-ETR2). In contrast, signalling through TRAIL-R1 by receptor-selective ligands or certain agonistic antibodies, such as mapatumumab (HGS-ETR1), occurs in the absence of cross-linking. These results further highlight important differences in apoptotic signalling triggered through TRAIL-R1 and TRAIL-R2 in primary tumour cells. Such information is clearly important for the rational optimisation of TRAIL therapy in primary lymphoid malignancies, such as CLL.
Assuntos
Apoptose/efeitos dos fármacos , Efrinas/fisiologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais Murinos/uso terapêutico , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas , Interações Medicamentosas , Feminino , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , PanobinostatRESUMO
Magnetic supports are tested for use in batch affinity capture of proteins. Two types of magnetic polymer composites were used for solid phase synthesis and for the batch affinity chromatography of folate binding protein from a protein mixture. Gly-Gly-L-Methotrexate as well as other analogs were synthesized on magnetic supports consisting of either polyoxyalkyleneamine grafted onto polystyrene beads or a copolymer of polyethylene glycol dimethylacrylamide (PEGA). Both supports incorporated within their matrix sub-micron particles of paramagnetic magnetite. The peptide-methotrexate analogs were attached to the magnetic supports via a photocleavable linker. The bound methotrexate-peptide analogs were equilibrated with a protein mixture consisting of bovine albumin, chicken albumin, folate binding protein, lysozyme, lactoferrin and lactoperoxidase precursor in phosphate buffered saline (PBS) and then after magnetically separating and washing the supports of any unbound components the bound protein was removed either through the photocleavage of the tethered methotrexate-peptide ligand or via exchange with soluble methotrexate. In all cases, the photocleavage or exchange with soluble methotrexate released folate binding protein as the major affinity captured protein. Of the two magnetic supports tested, the PEGA based support was found to be superior to the polyoxyalkyleneamine grafted polystyrene support and comparable to beaded agarose in releasing bound folate binding protein. Of the two methods for removing bound protein, photocleavage of the covalently attached ligand was found to release exclusively folate binding protein as opposed to exchange with soluble methotrexate which released residual amounts of the non-specifically bound proteins bovine and chicken albumin, in addition to folate binding protein. Thus, use of the PEGA based magnetic support in conjunction with a photocleavable linker should help facilitate the automation of multiple parallel affinity chromatography for proteomics applications.