RESUMEN
The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these beta-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11-70), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2-3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11-70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2-/-cgamma-/- mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces beta-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/terapia , Quimiocinas CC/fisiología , Inmunidad Innata , Familia de Multigenes/inmunología , Trasplante Heterólogo/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales de Origen Murino , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/biosíntesis , Quimiocinas CC/genética , Proteínas del Sistema Complemento/fisiología , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunidad Innata/genética , Masculino , Ratones , Ratones Desnudos , Familia de Multigenes/genética , ARN Mensajero/biosíntesis , RituximabRESUMEN
BACKGROUND AND OBJECTIVES: We have set up a murine B lymphoma model stably expressing human CD20 and homing in lymph nodes in immunocompetent mice to study the mechanism of action of rituximab. DESIGN AND METHODS: The B lymphoma line 38C13 was stably transduced with the human CD20 cDNA by retroviral infection and injected into syngeneic mice. RESULTS: The transduced 38C13-CD20(+) cells stably expressed human CD20 on 100% of cells. Rituximab alone did not inhibit 38C13-CD20+ cell growth but relocalized the human CD20 into lipid rafts and induced complement-mediated lysis in vitro. Inoculation of 4x10(3) 38C13-CD20(+) intravenously into syngeneic mice led to the development of tumor masses in the spleen, bone marrow and lymph nodes, detectable from day 15 by polymerase chain reaction (PCR) analysis, and with a median survival of 21-24 days. Treatment with 250 mg rituximab i.p. given 1-10 days after tumor inoculation cured 100% of animals, with disappearance of tumor documented by immunohistochemistry and PCR analysis. Depletion of both NK cells and neutrophils did not affect the therapeutic activity of rituximab in vivo. Similarly, removal of phagocytic macrophages using clodronate-liposomes did not modify the capacity of rituximab to control tumor growth. In contrast, the protective activity of the antibody was completely abolished after complement depletion with cobra venom factor. Complement was also required when cells were inoculated subcutaneously in nude mice. INTERPRETATION AND CONCLUSIONS: These data demonstrate that complement is required for the therapeutic activity of rituximab in vivo in a murine model of B-cell lymphoma, independently of its localization in lymph nodes or subcutaneously.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Proteínas del Sistema Complemento/fisiología , Ganglios Linfáticos/patología , Linfoma de Células B/patología , Animales , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Rituximab , Transducción GenéticaRESUMEN
The therapeutic unconjugated anti-CD20 Mab rituximab is used for the treatment of B-non-Hodgkin's lymphomas. We have studied the direct biological effects, signalling and gene expression profiles induced by rituximab in two human B-lymphoma cell lines, DHL4 and BJAB, using microarray, quantitative PCR and gel shift analysis. Rituximab alone inhibited thymidine uptake and induced homotypic adhesion in DHL4 only, but not BJAB. Analysis of Affymetrix microchips carrying probes for about 10,000 human cDNAs, allowed us to identify 16 genes in DHL4 and 12 in BJAB induced by rituximab at 4 h. Eleven and seven of these genes were specific for DHL4 and BJAB, respectively; whereas the remaining five were up-regulated in both cell lines. Mean induction ranged from 2- to 16-fold. Real time PCR analysis allowed us to confirm up-regulation of all genes identified, except one in BJAB. Time course of induction of eight genes was studied, showing peak induction in most cases at 4 h. The up-regulation of 5/5 genes was also observed with the F(ab')(2) fragment of rituximab. Analysis of three further B-cell lymphoma lines showed that gene induction is not restricted to BJAB and DHL4. Finally, we show that rituximab alone can induce AP1 activation in both cell lines and provide evidence that the ERK1/2 pathway is involved in the rituximab-mediated up-regulation of gene expression. These data demonstrate that rituximab alone has direct signalling capacity in different B-lymphoma lines, inducing distinct but overlapping sets of genes which may play a role in the biological and/or therapeutic effect of the antibody.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/biosíntesis , Antineoplásicos/farmacología , Apoptosis , Adhesión Celular , Línea Celular Tumoral , Cartilla de ADN/química , ADN Complementario/metabolismo , Daclizumab , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoglobulina G/farmacología , Etiquetado Corte-Fin in Situ , Linfoma/patología , Linfoma de Células B/patología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN/metabolismo , ARN Complementario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rituximab , Timidina/metabolismo , Timidina/farmacología , Factores de Tiempo , Factor de Transcripción AP-1/biosíntesis , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20(+) lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20(+) cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa(-/-)). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.