RESUMO
Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rß (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.
RESUMO
Although CD3-bispecific antibodies have shown promising activity in the treatment of hematological cancers, insufficient T-cell costimulation may limit long-term responses. Immunomodulatory drugs (IMiDs), routinely used in treating multiple myeloma, possess pleiotropic antimyeloma properties and have been described to enhance T-cell responses similar to costimulatory signaling and may therefore have synergistic effects when combined with T-cell bispecifics. In this report, we demonstrate that IMiDs substantially enhance tumor cell killing induced by CD3 bispecifics and increase CD8+ T-cell proliferation and expansion. We further show that the beneficial effects of IMiDs on T-cell function and expansion are mediated by enhanced IL2 production by CD4+ T cells. Our studies provide mechanistic insight into the costimulatory properties of IMiDs and support combination treatments with T-cell agonist therapies in a broad spectrum of indications.
Assuntos
Anticorpos Biespecíficos , Humanos , Anticorpos Biespecíficos/farmacologia , Agentes de Imunomodulação , Interleucina-2/farmacologia , Complexo CD3 , Linfócitos T CD8-PositivosRESUMO
Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with â¼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Neoplasias Ovarianas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/patologiaRESUMO
Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.
Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos , Antineoplásicos Imunológicos/imunologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Biespecíficos/química , Antineoplásicos Imunológicos/química , Complexo CD3/química , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Macaca fascicularis , Receptor ErbB-2/químicaRESUMO
T cell-retargeting therapies have transformed the therapeutic landscape of oncology. Regardless of the modality, T cell activating therapies are commonly accompanied by systemic cytokine release, which can progress to deadly cytokine release syndrome (CRS). Because of incomplete mechanistic understanding of the relationship between T cell activation and systemic cytokine release, optimal toxicity management that retains full therapeutic potential remains unclear. Here, we report the cell type-specific cellular mechanisms that link CD3 bispecific antibody-mediated killing to toxic cytokine release. The immunologic cascade is initiated by T cell triggering, whereas monocytes and macrophages are the primary source of systemic toxic cytokine release. We demonstrate that T cell-generated tumor necrosis factor-α (TNF-α) is the primary mechanism mediating monocyte activation and systemic cytokine release after CD3 bispecific treatment. Prevention of TNF-α release is sufficient to impair systemic release of monocyte cytokines without affecting antitumor efficacy. Systemic cytokine release is only observed upon initial exposure to CD3 bispecific antibody not subsequent doses, indicating a biological distinction between doses. Despite impaired cytokine release after second exposure, T cell cytotoxicity remained unaffected, demonstrating that cytolytic activity of T cells can be achieved in the absence of cytokine release. The mechanistic uncoupling of toxic cytokines and T cell cytolytic activity in the context of CD3 bispecifics provides a biological rationale to clinically explore preventative treatment approaches to mitigate toxicity.
Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Linfócitos T Citotóxicos/imunologia , Animais , Humanos , Macrófagos/metabolismo , Camundongos Transgênicos , Monócitos/metabolismo , Receptor ErbB-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A primary barrier to the success of T cell-recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell-dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab-immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell-directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.
Assuntos
Afinidade de Anticorpos/imunologia , Complexo CD3/imunologia , Citotoxicidade Imunológica , Receptor ErbB-2/imunologia , Anticorpos Biespecíficos/imunologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Ativação Linfocitária/imunologia , Ligação ProteicaRESUMO
PURPOSE: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. EXPERIMENTAL DESIGN: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. RESULTS: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. CONCLUSIONS: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.
Assuntos
Anticorpos Biespecíficos/farmacologia , Complexo CD3/antagonistas & inibidores , Quimiocinas/metabolismo , Interferon gama/metabolismo , Neoplasias/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptores CXCR3/metabolismo , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/patologia , Transdução de Sinais , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Complexo CD3/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Afinidade de Anticorpos , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/patologia , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Epitopos , Sinapses Imunológicas/fisiologia , Mieloma Múltiplo/tratamento farmacológico , Receptores Fc/imunologia , Linfócitos T/imunologia , Animais , Citocinas/metabolismo , Humanos , Antígenos Comuns de Leucócito/fisiologia , Ativação Linfocitária , Macaca fascicularis , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Receptor de Morte Celular Programada 1/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Fc/análiseRESUMO
Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG1, IgG2 and IgG4 thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.
Assuntos
Anticorpos Biespecíficos/biossíntese , Engenharia de Proteínas/métodos , Animais , Humanos , Imunoglobulina GRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Fucose/imunologia , Células Matadoras Naturais/imunologia , Receptores de IgG/imunologia , Actinas/metabolismo , Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/imunologia , Degranulação Celular/imunologia , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Transdução de Sinais/imunologia , Trastuzumab/química , Trastuzumab/imunologiaRESUMO
Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.
Assuntos
Anticorpos Biespecíficos/imunologia , Ativação Linfocitária , Receptor ErbB-2/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Ratos , Ratos Sprague-Dawley , TrastuzumabRESUMO
OBJECTIVE: ErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma. METHODS: A tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar. RESULTS: While ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro. CONCLUSIONS: These results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer.
Assuntos
Cistadenocarcinoma Seroso/enzimologia , Receptores ErbB/análise , Neoplasias Ovarianas/enzimologia , Adulto , Idoso , Proliferação de Células , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Receptores ErbB/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Isoenzimas/análise , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Receptor ErbB-4 , Análise Serial de TecidosRESUMO
First generation antibody drugs recognize the cancer cell, slow down the signaling of cell growth and activate the defense response. Second generation antibody drugs contain conjugated cytotoxic agents that are activated upon entry into the cancer cell. Trastuzumab has become established among the first generation antibody drugs utilized in breast cancer therapy, and its derivative trastuzumab-DM1 is the first antibody-drug conjugate currently in clinical trials for breast cancer. Trastuzumab acts as an antibody and transports into the cancer cell the cytotoxic agent DM1, which becomes activated there. Targeted cytotoxic drugs are under development for the treatment of many different types of cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Masculino , TrastuzumabRESUMO
Trastuzumab (Herceptin(®)) is currently used as a treatment for patients whose breast tumors overexpress HER2/ErbB2. Trastuzumab-DM1 (T-DM1, trastuzumab emtansine) is designed to combine the clinical benefits of trastuzumab with a potent microtubule-disrupting drug, DM1 (a maytansine derivative). Currently T-DM1 is being tested in multiple clinical trials. The mechanisms of action for trastuzumab include inhibition of PI3K/AKT signaling pathway, inhibition of HER-2 shedding and Fcγ receptor mediated engagement of immune cells, which may result in antibody-dependent cellular cytotoxicity (ADCC). Here we report that T-DM1 retains the mechanisms of action of unconjugated trastuzumab and is active against lapatinib resistant cell lines and tumors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunotoxinas/uso terapêutico , Maitansina/análogos & derivados , Quinazolinas/efeitos adversos , Receptor ErbB-2/antagonistas & inibidores , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados , Lapatinib , Maitansina/uso terapêutico , Camundongos , Camundongos Nus , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , TrastuzumabRESUMO
The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcgammaRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function-enhanced antibodies for solid tumor therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Fucose/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , Receptores de IgG/biossíntese , Receptores de IgG/genética , Receptores de IgG/metabolismo , TrastuzumabRESUMO
Herceptin (trastuzumab) is the backbone of HER2-directed breast cancer therapy and benefits patients in both the adjuvant and metastatic settings. Here, we describe a mechanism of action for trastuzumab whereby antibody treatment disrupts ligand-independent HER2/HER3 interactions in HER2-amplified cells. The kinetics of dissociation parallels HER3 dephosphorylation and uncoupling from PI3K activity, leading to downregulation of proximal and distal AKT signaling, and correlates with the antiproliferative effects of trastuzumab. A selective and potent PI3K inhibitor, GDC-0941, is highly efficacious both in combination with trastuzumab and in the treatment of trastuzumab-resistant cells and tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Indazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Sulfonamidas/farmacologia , Animais , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Ligantes , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , TrastuzumabRESUMO
Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.
Assuntos
Proteínas Angiogênicas/metabolismo , Endotélio Vascular/metabolismo , Neovascularização Fisiológica , Neuregulina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Angiogênicas/farmacologia , Hipóxia Celular , Movimento Celular , Endotélio Vascular/química , Endotélio Vascular/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neuregulina-1/genética , Neuregulina-1/farmacologia , Comunicação Parácrina , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Cordão Umbilical/citologia , Regulação para Cima , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: The epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) has shown antitumor activity in clinical trials against cancers, such as non-small cell lung cancer and head and neck squamous cell carcinoma (HNSCC). Research on non-small cell lung cancer has elucidated factors that may predict response to gefitinib. Less is known about molecular markers that may predict response to gefitinib in HNSCC patients. EXPERIMENTAL DESIGN: We analyzed possible associations of responsiveness to gefitinib with molecular markers of the EGFR/ErbB receptor family signaling pathway using 10 established HNSCC lines in vitro. IC50 of gefitinib sensitivity was determined using clonogenic survival assays. ErbB signaling was assessed by Western and real-time reverse transcription-PCR analyses of EGFR, ErbB2, ErbB3, and ErbB4 expression levels as well as by phosphorylation analysis of pEGFR, pErbB2, pErbB3, pAkt, and pErk. EGFR sequences encoding kinase domain and EGFR gene copy numbers were determined by cDNA sequencing and real-time PCR, respectively. Finally, responsiveness to gefitinib was compared with responsiveness to the anti-EGFR antibody cetuximab (Erbitux). RESULTS: Expression levels of pErbB2 (P = 0.02) and total ErbB3 protein (P = 0.02) associated with resistance to gefitinib. Combining gefitinib with pertuzumab (Omnitarg), an antibody targeting ErbB2 heterodimerization, provided additional growth-inhibitory effect over gefitinib alone on relatively gefitinib-resistant HNSCC cell lines. The same markers did not predict resistance to cetuximab. In contrast, a similar trend suggesting association between EGFR gene copy number and drug sensitivity was observed for both gefitinib (P = 0.0498) and cetuximab (P = 0.053). No activating EGFR mutations were identified. CONCLUSIONS: EGFR amplification may predict sensitivity to gefitinib in HNSCC. However, other EGFR/ErbB receptor family members than EGFR may contribute to resistance to gefitinib. ErbB2 and ErbB3 may have potential as predictive markers and as therapeutic targets for combination therapy in treatment of HNSCC with gefitinib.
Assuntos
Carcinoma de Células Escamosas/genética , Receptores ErbB/antagonistas & inibidores , Amplificação de Genes , Neoplasias de Cabeça e Pescoço/genética , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Cetuximab , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Gefitinibe , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Masculino , Quinazolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Análise de Sequência de DNA , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-alpha converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by gamma-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.