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1.
Haematologica ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296574

RESUMO

CD38 is expressed in several types of non-Hodgkin lymphoma and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory multiple myeloma. It has also demonstrated clinical activity in Waldenstrom macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma, follicular lymphoma and diffuse large B cell lymphoma, as monotherapy or in combination with standard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-dependent cell phagocytosis in all lymphoma subtypes. In the presence of human serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratumumab fully penetrated a 3D lymphoma organoid and decreased organoid volume. In vivo, daratumumab completely prevents tumor outgrowth in models of mantle cell and follicular lymphoma, and shows comparable activity to rituximab in a disseminated in vivo model of blastic mantle cell lymphoma. Moreover, daratumumab improves overall survival in a mouse model of transformed CD20dim follicular lymphoma, where rituximab showed limited activity. Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in mantle cell and follicular lymphoma xenografts. Furthermore, in a patient-derived diffuse large B cell lymphoma xenograft model, daratumumab anti-tumor activity was comparable to R-CHOP and the addition of daratumumab to either CHOP or R-CHOP led to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant further clinical development.

2.
J Immunol ; 197(3): 807-13, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27316683

RESUMO

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Mieloma Múltiplo/imunologia , Receptores de IgG/imunologia , ADP-Ribosil Ciclase 1 , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/efeitos dos fármacos , Células Tumorais Cultivadas
3.
Immunol Rev ; 270(1): 95-112, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26864107

RESUMO

CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hematological tumors, and shows especially broad and high expression levels in plasma cell tumors such as multiple myeloma (MM). Together, this triggered the development of various therapeutic CD38 antibodies, including daratumumab, isatuximab, and MOR202. Daratumumab binds a unique CD38 epitope and showed strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity, and immunomodulatory activity. CD38-targeting antibodies have a favorable toxicity profile in patients, and early clinical data show a marked activity in MM, while studies in other hematological malignancies are ongoing. Daratumumab has single agent activity and a limited toxicity profile, allowing favorable combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases.


Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos Clínicos como Assunto , Citotoxicidade Imunológica , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/metabolismo , Humanos , Imunomodulação/efeitos dos fármacos , Ligação Proteica , Recidiva , Resultado do Tratamento
4.
Haematologica ; 101(5): 616-25, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26858358

RESUMO

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Imunoterapia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/imunologia , Animais , Citocinas/biossíntese , Citotoxicidade Imunológica , Modelos Animais de Doenças , Citometria de Fluxo , Expressão Gênica , Técnicas de Transferência de Genes , Genes Transgênicos Suicidas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplante , Transdução Genética , Carga Tumoral/genética , Carga Tumoral/imunologia
5.
Transfusion ; 55(6 Pt 2): 1555-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25988285

RESUMO

BACKGROUND: Monoclonal antibodies (MoAbs) are increasingly integrated in the standard of care. The notion that therapeutic MoAbs can interfere with clinical laboratory tests is an emerging concern that requires immediate recognition and the development of appropriate solutions. Here, we describe that treatment of multiple myeloma patients with daratumumab, a novel anti-CD38 MoAb, resulted in false-positive indirect antiglobulin tests (IATs) for all patients for 2 to 6 months after infusion. This precluded the correct identification of irregular blood group antibodies for patients requiring blood transfusion. STUDY DESIGN AND METHODS: The IAT was performed using three- and 11-donor-cell panels. Interference of daratumumab and three other anti-CD38 MoAbs was studied using fresh-frozen plasma spiked with different MoAb concentrations. Additionally it was tested whether two potentially neutralizing agents, anti-idiotype antibody and recombinant soluble CD38 (sCD38) extracellular domain, were able to inhibit the interference. RESULTS: The CD38 MoAbs caused agglutination in the IAT in a dose-dependent manner. Addition of an excess of anti-idiotype antibodies or sCD38 protein to the test abrogated CD38 MoAb interference and successfully restored irregular antibody screening and identification. DISCUSSION: CD38 MoAb therapy causes false-positive results in the IAT. The reliability of the test could be restored by adding a neutralizing agent against the CD38 MoAb to the patient's plasma. This study emphasizes that during drug development, targeted therapeutics should be investigated for potential interference with laboratory tests. Clinical laboratories should be informed when patients receive MoAb treatments and matched laboratory tests to prevent interference should be employed.


Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Tipagem e Reações Cruzadas Sanguíneas , Transfusão de Sangue , ADP-Ribosil Ciclase 1/imunologia , Idoso , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Tipagem e Reações Cruzadas Sanguíneas/normas , Transfusão de Sangue/métodos , Teste de Coombs/normas , Reações Cruzadas , Reações Falso-Positivas , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Testes Sorológicos
6.
MAbs ; 7(2): 311-21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25760767

RESUMO

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Citofagocitose/efeitos dos fármacos , Linfoma/tratamento farmacológico , Macrófagos/imunologia , Mieloma Múltiplo/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Humanos , Linfoma/imunologia , Linfoma/patologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Haematologica ; 100(2): 263-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25510242

RESUMO

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines, but it did significantly augment daratumumab-induced myeloma cell lysis. Also in an ex vivo setting, IPH2102 synergistically improved daratumumab-dependent lysis of primary myeloma cells in bone marrow mononuclear cells (n=21), especially in patients carrying the FcγRIIIa-158F allele or the FcγRIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcγRIIIa-158V allele or the FcγRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citotoxicidade Imunológica/imunologia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Receptores KIR/imunologia , Western Blotting , Proliferação de Células , Células Cultivadas , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lenalidomida , Mieloma Múltiplo/imunologia , Polimorfismo Genético/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de IgG/genética , Receptores KIR/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Talidomida/administração & dosagem , Talidomida/análogos & derivados
8.
Neoplasia ; 14(3): 190-205, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22496619

RESUMO

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPß-LIP. Thus, siRNA-mediated knockdown of C/EBPß led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPß-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas ras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/genética , Antineoplásicos/farmacologia , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética , Proteínas ras/genética
10.
J Immunol ; 187(6): 3383-90, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21832160

RESUMO

Ab-dependent cellular cytotoxicity (ADCC) is recognized as a prominent cytotoxic mechanism for therapeutic mAbs in vitro. However, the contribution of ADCC to in vivo efficacy, particularly for treatment of solid tumors, is still poorly understood. For zalutumumab, a therapeutic epidermal growth factor receptor (EGFR)-specific mAb currently in clinical development, previous studies have indicated signaling inhibition and ADCC induction as important therapeutic mechanisms of action. To investigate the in vivo role of ADCC, a panel of EGFR-specific mAbs lacking specific functionalities was generated. By comparing zalutumumab with mAb 018, an EGFR-specific mAb that induced ADCC with similar potency, but did not inhibit signaling, we observed that ADCC alone was insufficient for efficacy against established A431 xenografts. Interestingly, however, both zalutumumab and mAb 018 prevented tumor formation upon early treatment in this model. Zalutumumab and mAb 018 also completely prevented outgrowth of lung metastases, in A431 and MDA-MB-231-luc-D3H2LN experimental metastasis models, already when given at nonsaturating doses. Finally, tumor growth of mutant KRAS-expressing A431 tumor cells, which were resistant to EGFR signaling inhibition, was completely prevented by early treatment with zalutumumab and mAb 018, whereas ADCC-crippled N297Q-mutated variants of both mAbs did not show any inhibitory effects. In conclusion, ADCC induction by EGFR-specific mAbs represents an important mechanism of action in preventing tumor outgrowth or metastasis in vivo, even of cancers insensitive to EGFR signaling inhibition.


Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/imunologia , Receptores ErbB/antagonistas & inibidores , Neoplasias Experimentais/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Separação Celular , Receptores ErbB/imunologia , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCID , Neoplasias Experimentais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Sci ; 102(10): 1761-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21718386

RESUMO

Monoclonal antibodies (mAb) against variant III of epidermal growth factor receptor (EGFRvIII) hold promise for improving tumor selectivity of EGFR-targeted therapy. Here, we compared Fc-mediated effector functions of three mAb against EGFRvIII (MR1-1, ch806, 13.1.2) with those of zalutumumab, a high affinity EGFR mAb in advanced clinical trials. MR1-1 and ch806 demonstrated preferential and 13.1.2 exclusive binding to EGFRvIII, in contrast to zalutumumab, which bound both wild-type and EGFRvIII. All four human IgG1κ mAb mediated antibody-dependent cellular cytotoxicity (ADCC) of EGFRvIII-expressing cells with mononuclear cells and isolated monocytes, while only zalutumumab in addition triggered ADCC by polymorphonuclear cells. Interestingly, combinations of zalutumumab and EGFRvIII mAb specifically mediated complement-dependent cytotoxicity (CDC) of EGFRvIII-transfected but not wild-type cells. Moreover, EGFRvIII-specific CDC was significantly enhanced when zalutumumab was combined with a Fc-engineered variant of MR1-1 (K326A/E333A). These observations confirm the immunotherapeutic potential of antibody combinations against EGFR, and demonstrate that tumor selectivity can be improved by combining therapeutic EGFR mAb with an antibody against EGFRvIII.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Mapeamento de Epitopos , Células HEK293 , Humanos , Neutrófilos/imunologia
13.
J Immunol ; 184(1): 512-20, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19949082

RESUMO

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.


Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais Humanizados , Linhagem da Célula , Citometria de Fluxo , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Camundongos , Monócitos/imunologia , Neutrófilos/imunologia , Panitumumabe
14.
Biotechnol J ; 3(9-10): 1157-71, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18702090

RESUMO

Monoclonal antibodies represent a major and increasingly important category of biotechnology products for the treatment of human diseases. The state-of-the-art of antibody technology has evolved to the point where therapeutic monoclonal antibodies, that are practically indistinguishable from antibodies induced in humans, are routinely generated. We depict how our science-based approach can be used to further improve the efficacy of antibody therapeutics, illustrated by the development of three monoclonal antibodies for various cancer indications: zanolimumab (directed against CD4), ofatumumab (directed against CD20) and zalutumumab (directed against epidermal growth factor receptor).


Assuntos
Anticorpos Monoclonais/uso terapêutico , Modelos Biológicos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Humanos , Resultado do Tratamento
15.
Cancer Res ; 68(13): 4998-5003, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18593896

RESUMO

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.


Assuntos
Anticorpos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas do Sistema Complemento/fisiologia , Receptores ErbB/imunologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Células CACO-2 , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Cetuximab , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Mapeamento de Epitopos , Humanos , Imunoglobulina A/administração & dosagem , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Modelos Biológicos , Neoplasias/imunologia , Panitumumabe , Células Tumorais Cultivadas
16.
Blood ; 112(6): 2390-9, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18566325

RESUMO

Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which fucosylation particularly affects NK cell-mediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG(1) monoclonal antibody against epidermal growth factor receptor in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation, and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance mononuclear cell-mediated antibody-mediated cellular cytotoxicity (ADCC). In contrast, polymorphonuclear cells were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type.


Assuntos
Anticorpos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Fucose/imunologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Anticorpos/imunologia , Linhagem Celular , Fucose/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo
17.
Proc Natl Acad Sci U S A ; 105(16): 6109-14, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18427122

RESUMO

The epidermal growth factor receptor (EGFR) activates cellular pathways controlling cell proliferation, differentiation, migration, and survival. It thus represents a valid therapeutic target for treating solid cancers. Here, we used an electron microscopy-based technique (Protein Tomography) to study the structural rearrangement accompanying activation and inhibition of native, individual, EGFR molecules. Reconstructed tomograms (3D density maps) showed a level of detail that allowed individual domains to be discerned. Monomeric, resting EGFR ectodomains demonstrated large flexibility, and a number of distinct conformations were observed. In contrast, ligand-activated EGFR complexes were detected only as receptor dimers with ring-like conformations. Zalutumumab, a therapeutic inhibitory EGFR antibody directed against domain III, locked EGFR molecules into a very compact, inactive conformation. Biochemical analyses showed bivalent binding of zalutumumab to provide potent inhibition of EGFR signaling. The structure of EGFR-zalutumumab complexes on the cell surface visualized by Protein Tomography indicates that the cross-linking spatially separates the EGFR molecules' intracellular kinase domains to an extent that appears incompatible with the induction of signaling. These insights into the mechanisms of action of receptor inhibition may also apply to other cell-surface tyrosine kinase receptors of the ErbB family.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Animais , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Mapeamento de Epitopos , Receptores ErbB/genética , Humanos , Ligantes , Camundongos , Microscopia Eletrônica , Mutação , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos
18.
J Immunol ; 180(6): 4338-45, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18322248

RESUMO

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.


Assuntos
Anticorpos Antineoplásicos/fisiologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Mutação , Animais , Anticorpos Antineoplásicos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/genética , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/fisiologia , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Inibidores do Crescimento/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Mutação/imunologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/genética , Transdução de Sinais/imunologia
19.
Cancer Res ; 66(15): 7630-8, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16885363

RESUMO

The epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors and represents an attractive target for antibody therapy. Here, we describe the effect of receptor-mediated antibody internalization on the pharmacokinetics and dose-effect relationship of a therapeutic monoclonal antibody (mAb) against EGFR (2F8). This mAb was previously found therapeutically active in mouse tumor models by two dose-dependent mechanisms of action: blockade of ligand binding and induction of antibody-dependent cell-mediated cytotoxicity. In vitro studies showed 2F8 to be rapidly internalized by EGFR-overexpressing cells. In vivo, accelerated 2F8 clearance was observed in cynomolgus monkeys at low doses but not at high doses. This enhanced clearance seemed to be receptor dependent and was included in a pharmacokinetic model designed to explain its nonlinearity. Receptor-mediated clearance was also found to affect in situ antibody concentrations in tumor tissue. Ex vivo analyses of xenograft tumors of 2F8-treated nude mice revealed that relatively high antibody plasma concentrations were required for maximum EGFR saturation in high-EGFR-expressing human A431 tumors, in contrast to lower-EGFR-expressing human xenograft tumors. In summary, receptor-mediated antibody internalization and degradation provides a saturable route of clearance that significantly affects pharmacokinetics, particularly at low antibody doses. EGFR saturation in normal tissues does not predict saturation in tumor tissue as local antibody concentrations in EGFR-overexpressing tumors may be more rapidly reduced by antibody internalization. Consequently, antibody saturation of the receptor may be affected, thereby affecting the local mechanism of action.


Assuntos
Anticorpos Monoclonais/farmacocinética , Receptores ErbB/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Relação Dose-Resposta Imunológica , Receptores ErbB/biossíntese , Feminino , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
J Immunol ; 173(7): 4699-707, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15383606

RESUMO

Epidermal growth factor receptor (EGF-R) overexpression is common in a large number of solid tumors and represents a negative prognostic indicator. Overexpression of EGF-R is strongly tumor associated, and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1kappa) directed against EGF-R, developed using human Ig transgenic mice. mAb 2F8 effectively blocked binding of EGF and TGF-alpha to the EGF-R. At saturating concentrations, 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concentrations, associated with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo analysis of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor saturation and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we observed potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.


Assuntos
Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Receptores ErbB/fisiologia , Feminino , Inibidores do Crescimento/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/patologia , Neoplasias/prevenção & controle , Ligação Proteica , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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