RESUMEN
In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.
Asunto(s)
Antígenos CD20/análisis , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Receptor Notch1/genética , Histona Desacetilasa 1/análisis , Histona Desacetilasa 2/análisis , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunologíaRESUMEN
The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos/química , Linfoma de Burkitt/inmunología , Inmunoterapia/métodos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD20/química , Antígenos CD55/química , Antígenos CD59/química , Separación Celular , Clonación Molecular , Proteínas del Sistema Complemento , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Células Asesinas Naturales/citología , Macrófagos/citología , Ratones , Ratones SCID , Microscopía FluorescenteRESUMEN
Monoclonal antibodies (mAbs), successfully adopted in the treatment of several haematological malignancies, have proved almost ineffective in multiple myeloma (MM), because of the lack of an appropriate antigen for targeting and killing MM cells. Here, we demonstrate that PSGL1, the major ligand of P-Selectin, a marker of plasmacytic differentiation expressed at high levels on normal and neoplastic plasma cells, may represent a novel target for mAb-mediated MM immunotherapy. The primary effectors of mAb-induced cell-death, complement-mediated lysis (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), were investigated using U266B1 and LP1 cell-lines as models. Along with immunological mechanisms, the induction of apoptosis by PSGL1 cross-linking was assessed. The anti-PSGL1 murine mAb KPL1 induced death of MM cells in a dose- and time-dependent fashion and mediated a significant amount of ADCC. KPL1 alone mediated C1q deposition on target cells but proved unable to induce CDC due to inhibition of the lytic activity of complement by membrane complement regulators (mCRP) expressed on the cell surface. Consistently, CDC was induced by KPL1 upon mCRP blockage. Our results suggest a role for PSGL1 in MM humoral immunotherapy and support further in vivo studies assessing the effects of anti-PSGL1 mAbs on MM growth and interaction with the bone marrow microenvironment.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Glicoproteínas de Membrana/inmunología , Mieloma Múltiple/terapia , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Línea Celular Tumoral , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Humanos , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patologíaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its death receptors, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.
Asunto(s)
Fragmentos de Inmunoglobulinas/uso terapéutico , Leucemia/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Humanos , Leucemia/patología , Biblioteca de Péptidos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
Human umbilical vein endothelial cells (HUVEC) have previously been shown to synthesize the functional terminal pathway of complement based on the detection by radioimmunoassay of the terminal complement complex (TCC) on coincubated agarose beads. In addition, C7 secretion by these cells in amounts comparable to C3, as well as C7 mRNA, has recently been demonstrated. However, it has not been possible to detect C5-6 and C8 in the fluid phase, and only trace amounts of soluble C9. Against this background we examined whether mRNA for the remaining terminal complement factors was present in HUVEC. By the use of reverse transcription (RT)-polymerase chain reaction (PCR) and Northern blot the presence of mRNA for complement factors C5, C6, C8 and C9 was demonstrated.