RESUMO
mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.
Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Quimiocina CCL20/imunologia , Animais , Doenças Autoimunes/imunologia , Membrana Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Imunoglobulina G/imunologia , Inflamação/imunologia , Macaca fascicularis , Células Th17/imunologiaRESUMO
The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.
Assuntos
Interleucina-18/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Murinos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulinas/metabolismo , Interferon gama/biossíntese , Interleucina-18/administração & dosagem , Células Matadoras Naturais/metabolismo , Linfoma/tratamento farmacológico , Linfoma/imunologia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores Fc/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Rituximab , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/farmacologia , Antígenos CD20/imunologia , Antineoplásicos Alquilantes/administração & dosagem , Cloridrato de Bendamustina , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos SCID , Compostos de Mostarda Nitrogenada/administração & dosagem , Compostos de Mostarda Nitrogenada/farmacologia , Rituximab , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.
Assuntos
Glicoproteínas/biossíntese , Interferon gama/fisiologia , Interleucina-12/farmacologia , Monócitos/imunologia , Ativação Transcricional , Anticorpos/farmacologia , Células Cultivadas , Citocinas/farmacologia , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/antagonistas & inibidores , Interferon gama/imunologia , Interleucina-12/uso terapêutico , Células Matadoras Naturais/imunologia , Cinética , Monócitos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , RNA Mensageiro/biossíntese , Células Th1/imunologiaRESUMO
Interleukin (IL)-18 has profound antitumor activity when administered at high doses as a single agent for prolonged periods in BALB/c mice bearing late, well-established MOPC-315 tumors. Management with a qD x 27 schedule resulted in regression of tumors in all animals receiving 5 mg/kg/d. A protracted daily management regimen appears to be necessary to induce regression in this advanced tumor model. Biologic markers were assessed and appear to be potentially useful in evaluating the immunologic and antitumor activity of IL-18. The biomarkers of IL-18's immunologic activity include, but are not limited to, IL-1alpha, IL-2, IL-8, IL-10, IL-12, IL-13, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor. The profile of these circulating cytokines and their expression levels at baseline, and after IL-18 delivery, can be measured in the serum, as well as from splenocytes of mice or human peripheral blood mononuclear cells derived from either normal subjects or patients with cancer. We compared IL-18 and IL-12 alone or in combination for their ability to induce cytokine production and natural killer cytolytic activity. Our data support the notion that IL-18 induces a predominantly Th1 response, and that the mechanism of IL-18 activity differs from that of IL-12. The biologic activity of IL-18 management revealed by increases in serum levels of cytokines and enhancement of natural killer cytolytic activity will be useful as clinical trials initiate in 2002. Expression of interferon-gamma and granulocyte-macrophage colony-stimulating factor serum levels correlates directly over a broad dose escalation with the level of IL-18. Therefore, this provides a convenient pharmacodynamic reference to the biologic response to IL-18 that may serve to guide the conduct of clinical trials.
Assuntos
Citocinas/metabolismo , Imunidade Celular/efeitos dos fármacos , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Plasmocitoma/tratamento farmacológico , Células Th1/efeitos dos fármacos , Animais , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Injeções Intraperitoneais , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Recombinação Genética , Valores de Referência , Sensibilidade e Especificidade , Células Th1/imunologiaRESUMO
Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.