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Introducción: El rituximab, anticuerpo quimérico que reconoce la molécula CD20 humana, se ha utilizado en el tratamiento de diversos trastornos linfoproliferativos de células B. Para la selección de los potenciales beneficiarios del tratamiento con rituximab se han desarrollado técnicas que, mediante el uso de anticuerpos monoclonales, detectan la presencia del CD20 en los linfocitos de estos pacientes. Objetivo: Obtener y caracterizar un anticuerpo recombinante IgG1 de ratón específico para la molécula CD20 humana, que contenga las regiones variables del anticuerpo rituximab. Métodos: Para la expresión estable del anticuerpo recombinante se empleó la transducción lentiviral de células de embrión de riñón humano (HEK293). La caracterización inmunoquímica del anticuerpo se realizó por la técnica de Western Blot y su capacidad de reconocimiento de la molécula CD20 humana se evaluó por citometría de flujo e inmunohistoquímica. Resultados: Se obtuvo el anticuerpo 1F5 que reconoce, por citometría de flujo, la molécula CD20 en líneas celulares humanas de origen linfoide, así como en células de sangre periférica de humanos sanos y pacientes con trstornos linfoproliferativos de células B. Sin embargo, la técnica de inmunohistoquímica solo permitió detectar con este anticuerpo la molécula CD20 en tejidos frescos, no así en los embebidos en parafina. Conclusiones: Este trabajo sugiere las potencialidades del uso del anticuerpo 1F5 para las mediciones de la expresión de CD20 por citometría de flujo en pacientes con leucemias B o linfomas B avanzados en fase de leucemización. Esto complementaría los estudios para la selección apropiada de pacientes para el tratamiento con el rituximab(AU)
Introduction: Rituximab, chimeric antibody specific for human CD20 molecule, has been widely used in the treatment of several B-cell linfoproliferative disorders. For the selection of patients with the greatest potential to benefit from the therapy with rituximab, a number of techniques using monoclonal antibodies have been developed to detect the CD20 molecule. Objective: To obtain and to characterize a mouse IgG1 recombinant antibody, specific for human CD20, that contains the variable regions of rituximab. Methods: The lentiviral transduction of human embryonic kidney cells (HEK293) was used for the stable expression of the recombinant antibody. The immunochemical characterization of the antibody was performed by Western Blot and the recognition of CD20 was evaluated by immunohistochemistry and flow cytometry. Results: We generated the antibody 1F5, able to recognize by flow cytometry the CD20 molecule expressed on lymphoid human cell lines, as well as peripheral blood mononuclear cells from healthy donors and patients with B-cell lymphoproliferative disorders. However, 1F5 antibody detected the CD20 molecule on fresh tissues, but not on formalin-fixed paraffin embedded tissues,by immunohistochemistry. Conclusions: This work suggests the potential use of 1F5 antibody for the measurement of CD20 expression by flow cytometry in patients with B-cell leukemias or B-cell lymphomas in phase of leukemization. This could complement the studies to ensure the appropriate selection of patients for the treatment with rituximab(AU)
Assuntos
Humanos , Masculino , Feminino , Imunoglobulina G/análise , Seleção de Pacientes/ética , Rituximab/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Anticorpos/uso terapêutico , Formação de Anticorpos , Western Blotting/métodos , Antígenos CD20/análiseRESUMO
The GM3(Neu5Gc) ganglioside represents a tumor-specific antigen that is considered a promising target for cancer immunotherapy. We previously demonstrated that the humanized antibody 14F7hT, specific for this ganglioside, exhibited significant antitumor effects in preclinical hematological tumor models. As this antibody recognizes human tumor tissues from several origins, we addressed its potential effect on different tumor types. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies, in particular for human malignancies, is complicated by the absence in humans of functional cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH), the enzyme required for Neu5Gc sialic acid biosynthesis. Quantitative flow cytometry revealed the absence of surface GM3(Neu5Gc) in several human but also mouse cell lines, in the last case due to low expression of the enzyme. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. However, only transfection of the mouse Cmah gene into human SKOV3 and mouse 3LL cells induced a stable expression of GM3(Neu5Gc) on the cancer cell surface, resulting in effective models to evaluate the antitumor responses by 14F7hT in vitro and in vivo. This antibody exerted antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo antitumor effects on these Cmah-transfected non-hematological tumors from both mouse and human origin. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel anti-cancer drug.
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Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/farmacologia , Gangliosídeo G(M3)/imunologia , Oxigenases de Função Mista/imunologia , Neoplasias/tratamento farmacológico , Animais , Antígenos de Neoplasias/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista/química , Neoplasias/imunologia , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
Liposomes containing phosphatidylcholine have been widely used as adjuvants. Recently, we demonstrated that B-1 cells produce dipalmitoyl-phosphatidylcholine (DPPC)-specific IgM upon immunization of BALB/c mice with DPPC-liposomes encapsulating ovalbumin (OVA). Although this preparation enhanced the OVA-specific humoral response, the contribution of anti-DPPC antibodies to this effect was unclear. Here, we demonstrate that these antibodies are secreted by B-1 cells independently of the presence of OVA in the formulation. We also confirm that these antibodies are specific for phosphocholine. The anti-OVA humoral response was partially restored in B-1 cells-deficient BALB/xid mice by immunization with the liposomes opsonized with the serum total immunoglobulin (Ig) fraction containing anti-phosphocholine antibodies, generated in wild-type animals. This result could be related to the increased phagocytosis by peritoneal macrophages of the particles opsonized with the serum total Ig or IgM fractions, both containing anti-phosphocholine antibodies. In conclusion, in the present work, it has been demonstrated that phosphocholine-specific antibodies improve T-dependent antibody responses against OVA carried by DPPC-liposomes.
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Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.
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Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Gangliosídeo G(M3)/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Gangliosídeo G(M3)/imunologia , Humanos , Isoenxertos , Camundongos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologiaRESUMO
B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.
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Antígenos/imunologia , Subpopulações de Linfócitos B/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos , Antígenos/química , Subpopulações de Linfócitos B/metabolismo , Movimento Celular , Feminino , Imunização , Lipossomos , Camundongos , Ovalbumina/imunologia , Fosfatidilcolinas/química , Fosfatidilcolinas/imunologia , Baço/imunologiaRESUMO
Macrophages respond to endogenous and non-self stimuli acquiring the M1 or M2 phenotypes, corresponding to classical or alternative activation, respectively. The role of B-1 cells in the regulation of macrophage polarization through the secretion of interleukin (IL)-10 has been demonstrated. However, the influence of B-1 cells on macrophage phenotype induction by an immunogen that suppress their ability to secrete IL-10 has not been explored. Here, we studied the peritoneal macrophage pattern induced by liposomes comprised of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) carrying ovalbumin (OVA) (Lp DPPC/OVA), and the involvement of B-1 cells in macrophage polarization. Peritoneal cells from BALB/c, B-1 cells-deficient BALB/xid and C57BL/6 mice immunized with Lp DPPC/OVA and OVA in soluble form (PBS/OVA) were analyzed and stimulated or not in vitro with lipopolysaccharide (LPS). Peritoneal macrophages from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA showed an M2-like phenotype as evidenced by their high arginase activity without LPS stimulation. Upon stimulation, these macrophages were reprogrammable toward the M1 phenotype with the upregulation of nitric oxide (NO) and a decrease in IL-10 secretion. In addition, high IFN-γ levels were detected in the culture supernatant of peritoneal cells from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA. Nevertheless, still high levels of arginase activity and undetectable levels of IL-12 were found, indicating that the switch to a classical activation state was not complete. In the peritoneal cells from liposomes-immunized BALB/xid mice, levels of arginase activity, NO, and IL-6 were below those from wild type animals, but the last two products were restored upon adoptive transfer of B-1 cells, together with an increase in IFN-γ secretion. Summarizing, we have demonstrated that Lp DPPC/OVA induce an M2-like pattern in peritoneal macrophages reprogrammable to M1 phenotype after LPS stimulation, with the involvement of B-1 cells.
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Linfócitos B/imunologia , Colesterol/farmacologia , Lipossomos/farmacologia , Macrófagos Peritoneais/imunologia , Ovalbumina/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Transferência Adotiva , Animais , Arginase/biossíntese , Linfócitos B/transplante , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/farmacologia , Interferon gama/biossíntese , Interleucina-10/metabolismo , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Fenótipo , Fosfatidilcolinas/farmacologiaRESUMO
Psoriasis is a chronic inflammatory disease with a prevalence of approximately 2-3% in the general population. The majority of diagnosed patients have plaque psoriasis, and about 20% have moderate-to-severe disease. Itolizumab, a new monoclonal antibody specific for the CD6 molecule mainly expressed on T lymphocytes, has demonstrated to inhibit in vitro ligand-induced proliferation and pro-inflammatory cytokine production. We assessed the immunological and histopathological effect of the antibody using clinical samples taken from 26 patients with moderate-to-severe psoriasis included in a clinical trial. The precursor frequency of lymphocytes activated with anti-CD2/CD3/CD28 beads, as well as the number of interferon (IFN)-γ-secreting T cells after stimulation, were measured at different time points of the study. Serum cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. Additionally, lymphocyte infiltration and epidermis hyperplasia were studied in five patients. A significant reduction in T cell proliferation capacity and number of IFN-γ-producing T cells was found in treated patients. Serum levels of interleukin-6, tumor necrosis factor and IFN-γ showed an overall trend toward reduction. No anti-idiotypic antibody response was detected. A significant reduction in the epidermis hyperplasia was observed in analyzed patients. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.
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Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Psoríase/imunologia , Psoríase/terapia , Adulto , Idoso , Anticorpos Anti-Idiotípicos/sangue , Proliferação de Células , Citocinas/sangue , Feminino , Humanos , Interferon gama/biossíntese , Masculino , Pessoa de Meia-Idade , Psoríase/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Adulto JovemRESUMO
Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.
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Carcinogênese/metabolismo , Gangliosídeo G(M3)/metabolismo , Leucemia Linfoide/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Animais , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular Tumoral , Feminino , Gangliosídeo G(M3)/imunologia , Células HEK293 , Humanos , Leucemia Linfoide/imunologia , Camundongos , Camundongos Endogâmicos DBA , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismoRESUMO
The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.
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Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos CD20/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos/genética , Anticorpos Monoclonais Murinos/farmacologia , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/genética , Antígenos CD20/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Cricetinae , Cricetulus , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Humanos , Células K562 , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Engenharia de Proteínas/métodos , RituximabRESUMO
Leukopenia is a severe condition resulting from both pathological processes and some treatments, like chemotherapy in cancer patients. However, the activation of the patient immune system is required for the success of immunotherapeutic strategies, as cancer vaccines. In this regard, leukopenia constitutes a major hurdle to overcome, mainly due to the impairment of cytotoxic T lymphocyte (CTL) responses. Adjuvants are basic components of vaccine formulations, which might be useful to stimulate immunity under this immunosuppressed condition. To this aim, we tested the capacity of a novel nanoparticulated complex, very small size proteoliposomes (VSSP), to promote CTL even in a leukopenic scenario. Noteworthy, we observed that a VSSP-based OVA vaccine induced a normal antigen-specific CTL response in mice rendered leukopenia by the administration of high doses of the chemotherapeutic agent cyclophosphamide (CY), while under the same conditions the OVA antigen formulated in the TLR-3 agonist polyinosinic-polycytidylic acid (P(I:C)) was ineffective. Moreover, an appropriate combination of VSSP with the P(I:C) vaccine was able to restore the CD8(+) T cell effector function in leukopenic mice. VSSP induced not only a faster repopulation of immune cells in CY-receiving animals, but also enhanced the recovery of memory T lymphocytes and myeloid dendritic cells (DCs) while simultaneously abrogated the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Our results suggest that VSSP could be a particularly suitable immunomodulator to be used in CTL-promoting active immunotherapy strategies operating in severe immune compromised scenarios.