RESUMO
The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.
Assuntos
Anticorpos Biespecíficos , Neoplasias Pancreáticas , Animais , Camundongos , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Transdução de Sinais , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias PancreáticasRESUMO
Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical efficacy remains limited in some other cases. Pre-clinical and clinical trials have shown that combinations of antibodies that bind to the same target (homo-combinations) or to different targets (hetero-combinations) to mimic the polyclonal humoral immune response improve their therapeutic effects in cancer. The approval of the trastuzumab/pertuzumab combination for breast cancer and then of the ipilimumab/nivolumab combination for melanoma opened the way to novel antibody combinations or oligoclonal antibody mixtures as more effective biologics for cancer management. We found more than 300 phase II/III clinical trials on antibody combinations, with/without chemotherapy, radiotherapy, small molecules or vaccines, in the ClinicalTrials.gov database. Such combinations enhance the biological responses and bypass the resistance mechanisms observed with antibody monotherapy. Usually, such antibody combinations are administered sequentially as separate formulations. Combined formulations have also been developed in which separately produced antibodies are mixed before administration or are produced simultaneously in a single cell line or a single batch of different cell lines as a polyclonal master cell bank. The regulation, toxicity and injection sequence of these oligoclonal antibody mixtures still need to be addressed in order to optimize their delivery and their therapeutic effects.
RESUMO
Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes. Here, we found by immunohistochemistry that these three receptors were co-expressed in 11% of patients with pancreatic adenocarcinoma. We then developed gemcitabine-resistant pancreatic cancer cell models (SW-1990-GR and BxPC3-GR) and one patient-derived xenograft (PDX2846-GR) by successive exposure to increasing doses of gemcitabine. We showed that expression of EGFR, HER2 and HER3 was increased in these gemcitabine-resistant pancreatic cancer models, and that an antibody mixture against all three receptors inhibited tumor growth in mice and downregulated HER receptors. Finally, we demonstrated that the Pan-HER and gemcitabine combination has an additive effect in vitro and in mice xenografted with the gemcitabine-sensitive or resistant pancreatic models. The mixture of anti-EGFR, HER2 and HER3 antibodies is a good candidate therapeutic approach for gemcitabine-sensitive and -resistant pancreatic cancer.
Assuntos
Anticorpos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos Nus , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3- /HER4+ cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 Ab C6, which binds to a conformational epitope located on a.a. 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptor ErbB-4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Humanos , Espaço Intracelular/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neuregulina-1/farmacologia , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-4/imunologiaRESUMO
PURPOSE: The outcome of locally advanced cervical cancer (LACC) is dismal. Biomarkers are needed to individualize treatments and to improve patient outcomes. Here, we investigated whether coexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) could be an outcome prognostic biomarker, and whether targeting both EGFR and HER3 with a dual antibody (MEHD7945A) enhanced ionizing radiation (IR) efficacy. METHODS AND MATERIALS: Expression of EGFR and HER3 was evaluated by immunohistochemistry in cancer biopsies (n = 72 patients with LACC). The antitumor effects of the MEHD7945A and IR combotherapy were assessed in 2 EGFR- and HER3-positive cervical cancer cell lines (A431 and CaSki) and in A431 cell xenografts. The mechanisms involved in tumor cell radiosensitization were also studied. The interaction of MEHD7945A, IR, and cisplatin was evaluated using dose-response matrix data. RESULTS: EGFR and HER3 were coexpressed in only in 7 of the 22 biopsies of FIGO IVB cervix cancer. The median overall survival was 14.6 months and 23.1 months in patients with FIGO IVB tumors that coexpressed or did not coexpress EGFR and HER3, respectively. In mice xenografted with A431 (squamous cell carcinoma) cells, MEHD7945A significantly increased IR response by reducing tumor growth and increasing cleaved caspase-3 expression. In A431 and CaSki cells, the combotherapy increased DNA damage and cell death, particularly immunogenic cell death, and decreased survival by inhibiting the MAPK and AKT pathways. An additive effect was observed when IR, MEHD7945A, and cisplatin were combined. CONCLUSIONS: Targeting EGFR and HER3 with a specific dual antibody enhanced IR efficacy. These preliminary results and the prognostic value of EGFR and HER3 coexpression should be confirmed in a larger sample.
Assuntos
Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Receptor ErbB-3/imunologia , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/imunologia , Sobrevivência Celular/efeitos da radiação , Transformação Celular Neoplásica , Terapia Combinada , Dano ao DNA , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Imunoglobulina G/uso terapêutico , Camundongos , Pessoa de Meia-Idade , Receptor ErbB-3/metabolismo , Estudos Retrospectivos , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapiaRESUMO
BACKGROUND: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways. Here, we wanted to determine the mechanisms underlying the pro-apoptotic effect of 9F7-F11. METHODS: Anti-HER3 antibody-induced apoptosis was assessed by western blot, and by flow cytometry measurement of Annexin V/7-AAD-labelled tumor cells (BxPC3, MDA-MB-468 and DU145 cell lines). c-FLIP/ITCH interaction and subsequent degradation/ubiquitination were investigated by co-immunoprecipitation of ITCH-silenced vs scramble control cells. The relationship between ITCH-mediated c-FLIP degradation and antibody-induced apoptosis was examined by western blot and flow cytometry of tumor cells, after ITCH RNA interference or by pre-treatment with ITCH chemical inhibitor chlorimipramine (CI). RESULTS: Following incubation with 9F7-F11, cancer cell apoptosis occurs through activation of caspase-8, - 9 and - 3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Moreover we showed that ubiquitination and proteasomal degradation of the anti-apoptotic protein c-FLIP was mediated by USP8-regulated ITCH recruitment. This effect was abrogated by ITCH- and USP8-specific RNA interference (siRNA), or by the ITCH chemical inhibitor CI. Specifically, ITCH silencing or CI blocked 9F7-F11-induced caspase-8-mediated apoptosis of tumor cells, and restored c-FLIP expression. ITCH-silencing or CI concomitantly abrogated HER3-specific antibody-induced apoptosis of Annexin V/7-AAD-labelled BxPC3 cells. 9F7-F11 favored the extrinsic apoptosis pathway by inducing TRAIL-R2/DR5 upregulation and TRAIL expression that promoted the formation of death-inducing signaling complex (DISC), leading to caspase-8-mediated apoptosis. Incubation with 9F7-F11 also induced BID cleavage, BAX upregulation and BIM expression, which initiated the caspase-9/3-mediated mitochondrial death pathway. The anti-HER3 antibody pro-apoptotic effect occurred concomitantly with downregulation of the pro-survival proteins c-IAP2 and XIAP. CONCLUSIONS: The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.
Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Humanos , Transdução de SinaisRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by poor response to chemotherapy and radiotherapy due to the lack of efficient therapeutic tools and early diagnostic markers. We previously generated the nonligand competing anti-HER3 antibody 9F7-F11 that binds to pancreatic tumor cells and induces tumor regression in vivo in experimental models. Here, we asked whether coupling 9F7-F11 with a radiosensitizer, such as monomethylauristatin E (MMAE), by using the antibody-drug conjugate (ADC) technology could improve radiation therapy efficacy in PDAC. We found that the MMAE-based HER3 antibody-drug conjugate (HER3-ADC) was efficiently internalized in tumor cells, increased the fraction of cells arrested in G2/M, which is the most radiosensitive phase of the cell cycle, and promoted programmed cell death of irradiated HER3-positive pancreatic cancer cells (BxPC3 and HPAC cell lines). HER3-ADC decreased the clonogenic survival of irradiated cells by increasing DNA double-strand break formation (based on γH2AX level), and by modulating DNA damage repair. Tumor radiosensitization with HER3-ADC favored the inhibition of the AKT-induced survival pathway, together with more efficient caspase 3/PARP-mediated apoptosis. Incubation with HER3-ADC before irradiation synergistically reduced the phosphorylation of STAT3, which is involved in chemoradiation resistance. In vivo, the combination of HER3-ADC with radiation therapy increased the overall survival of mice harboring BxPC3, HPAC cell xenografts or patient-derived xenografts, and reduced proliferation (KI67-positive cells). Combining auristatin radiosensitizer delivery via an HER3-ADC with radiotherapy is a new promising therapeutic strategy in PDAC.
Assuntos
Carcinoma Ductal Pancreático/terapia , Imunoconjugados/administração & dosagem , Fatores Imunológicos/administração & dosagem , Neoplasias Pancreáticas/terapia , Animais , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Fatores Imunológicos/farmacologia , Camundongos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Neoplasias Pancreáticas/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fator de Transcrição STAT3/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical effectiveness remains limited in some cases. Associations of antibodies binding to the same target (homo-combination) or to several different targets (hetero-combination), thereby mimicking a polyclonal humoral immune response, have demonstrated a therapeutic improvement in pre-clinical and clinical trials, mainly in the field of oncology and infectious diseases. The combinations increase the efficacy of the biological responses and override resistance mechanisms observed with antibody monotherapy. The most common method of formulating and administering antibody combinations is a separate formulation, with sequential injection of each antibody as individual drug substance. Alternatively, combined formulations are developed where the separately-produced antibodies are mixed before administration or produced simultaneously by a single cell line, or a mixture of cell lines as a polyclonal master cell bank. The regulation, the toxicity and the injection sequence of these oligoclonal antibody-based mixtures remain points to be clarified and optimized for a better therapeutic effect.
TITLE: Imiter la réponse immunitaire humorale polyclonale - De l'association de deux anticorps monoclonaux aux productions oligoclonales. ABSTRACT: Les anticorps monoclonaux ont révolutionné le traitement de nombreuses maladies mais leur efficacité clinique reste limitée dans certains cas. Des associations d'anticorps se liant à une même cible (homo-combinaisons) ou à plusieurs cibles différentes (hétéro-combinaisons), mimant ainsi une réponse immunitaire humorale polyclonale, ont conduit à une amélioration thérapeutique dans des essais précliniques et cliniques, essentiellement en cancérologie et en infectiologie. Ces combinaisons augmentent l'efficacité des réponses biologiques et court-circuitent les mécanismes de résistances observés lors d'une monothérapie par anticorps. Le procédé de formulation et d'administration des combinaisons d'anticorps le plus fréquent est une formulation séparée, avec injection séquentielle de chaque anticorps « principe actif ¼. Alternativement, se développent des formulations combinées, où les anticorps produits séparément sont mélangés avant administration, ou produits simultanément par une lignée cellulaire unique ou un mélange de lignées cellulaires correspondant à une master-bank cellulaire polyclonale. La réglementation, la toxicité et la séquence d'injection des mélanges oligoclonaux restent des points à éclaircir et à optimiser pour un meilleur effet thérapeutique.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Imunidade Humoral , Imunoterapia/métodos , Mimetismo Molecular , Bandas Oligoclonais/administração & dosagem , Animais , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/fisiologia , Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/terapia , Combinação de Medicamentos , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/fisiologia , Mimetismo Molecular/efeitos dos fármacos , Mimetismo Molecular/imunologia , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
BACKGROUND: The irinotecan-induced phosphokinome changes in colorectal cancer (CRC) cells were used to guide the selection of targeted agents to be tested in combination with irinotecan. METHODS: Phosphokinome profiling with peptide arrays of tumour samples from nude mice xenografted with HT29 cells and treated or not with an effective dose of irinotecan was used to identify signalling pathways activated by irinotecan treatment. Then, drugs targeting these pathways were combined in vitro with irinotecan to test potential synergistic effect. The interactions between these drug combinations were assessed by a dose matrix approach. Confirmation of the most potential combination has been confirmed in vivo in xenografted mice. RESULTS: Irinotecan induced in vivo the activation of AKT and MEK1 phosphorylation. The dose matrix approach showed that BKM120 (PI3K inhibitor) and MEK162 (MEK inhibitor) are synergistic in vitro and in vivo with a cytostatic and cytotoxic effect, while combination of BKM120 and irinotecan or MEK162 and irinotecan are only additive or even antagonistic. However, the triple combination of SN38, BKM120 and MEK162 showed a better synergistic effect that BKM120 and MEK162, indicating that the cells need to inhibit both AKT and ERK pathways to become more sensitive to irinotecan-based chemotherapies. CONCLUSION: Analysis of chemotherapy-induced phosphokinome changes helps to elucidate the mechanisms of drug resistance and to guide the selection of targets for combination therapies with synergistic activity.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Aminopiridinas/administração & dosagem , Aminopiridinas/efeitos adversos , Animais , Benzimidazóis/administração & dosagem , Benzimidazóis/efeitos adversos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HT29 , Humanos , Irinotecano , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Terapia de Alvo Molecular , Morfolinas/administração & dosagem , Morfolinas/efeitos adversos , Inibidores de Proteínas Quinases/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.
Assuntos
Anticorpos Monoclonais/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Carcinoma Ductal Pancreático/prevenção & controle , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neuregulina-1/antagonistas & inibidores , Neoplasias Pancreáticas/prevenção & controle , Receptor ErbB-3/antagonistas & inibidores , Animais , Apoptose , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Técnicas de Cocultura , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuregulina-1/imunologia , Neuregulina-1/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.
Assuntos
Antineoplásicos Imunológicos/química , Catepsina B/metabolismo , Imunoconjugados/química , Oligopeptídeos/química , Receptor ErbB-2/metabolismo , Trastuzumab/química , Antineoplásicos Imunológicos/síntese química , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética/métodos , Sistemas de Liberação de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Trastuzumab/farmacologiaRESUMO
Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Biomarcadores Tumorais/imunologia , Neoplasias/tratamento farmacológico , Neuregulina-1/genética , Receptor ErbB-3/imunologia , Células A549 , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Murinos/imunologia , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neuregulina-1/imunologia , Fosforilação , Ligação Proteica , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation. Clin Cancer Res; 23(11); 2806-16. ©2016 AACR.
Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
We characterized the mechanism of action of the neuregulin-non-competitive anti-HER3 therapeutic antibody 9F7-F11 that blocks the PI3K/AKT pathway, leading to cell cycle arrest and apoptosis in vitro and regression of pancreatic and breast cancer in vivo. We found that 9F7-F11 induces rapid HER3 down-regulation. Specifically, 9F7-F11-induced HER3 ubiquitination and degradation in pancreatic, breast and prostate cancer cell lines was driven mainly by the itchy E3 ubiquitin ligase (ITCH/AIP4). Overexpression of the ITCH/AIP4 inhibitor N4BP1 or small-interfering RNA-mediated knockdown of ITCH/AIP4 inhibited HER3 ubiquitination/degradation and PI3K/AKT signaling blockade induced by 9F7-F11. Moreover, 9F7-F11-mediated JNK1/2 phosphorylation led to ITCH/AIP4 activation and recruitment to HER3 for receptor ubiquitination and degradation. ITCH/AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference. Taken together, our results suggest that 9F7-F11-induced HER3 ubiquitination and degradation in cancer cells mainly occurs through JNK1/2-dependent ITCH/AIP4 activation.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor ErbB-3/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , UbiquitinaçãoAssuntos
Proteínas de Neoplasias/fisiologia , Receptor ErbB-3/fisiologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Receptores ErbB/fisiologia , Humanos , Terapia de Alvo Molecular , Família Multigênica , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
To improve treatment efficacy, we decided to simultaneously target HER1 and HER2 with trastuzumab and cetuximab. Following promising preclinical results, we conducted a phase 1-2 trial in advanced pancreatic cancer patients after first-line gemcitabine-based chemotherapy failure. In this single-arm, non-randomized, multicenter trial, patients received weekly cetuximab (400mg/m², then 250mg/m²). They were sequentially included in two trastuzumab dose levels: 3.0 or 4.0mg/kg, then 1.5 or 2.0mg/kg/weekly. Endpoints were the objective response rate, safety, progression-free (PFS) and overall survival (OS). During phase 1 (n=10 patients), toxicities were evenly distributed except for skin toxicities that frequently caused compliance issues. The higher dose level was defined as the trastuzumab recommended dose. During phase 2 (n=39 patients), toxicities were mainly cutaneous reactions and asthenia. No objective response was observed. Nine patients were stabilized but arrested treatment due to toxicity. Median PFS was 1.8 months (95%CI: 1.7-2.0 months) and median OS was 4.6 months (95%CI: 2.7-6.6 months). Both were positively correlated with skin toxicity severity (P=0.027 and P=0.001, respectively). Conventional phase 1 dose-escalation schedules are unsuitable for targeted therapies because most cutaneous toxicities are not considered dose-limiting toxicities. The compliance issues caused by skin toxicities were particularly detrimental because of the toxicity-response correlation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Cetuximab/administração & dosagem , Cetuximab/efeitos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Intervalo Livre de Doença , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias Pancreáticas/mortalidade , Receptor ErbB-2/antagonistas & inibidores , Terapia de Salvação/métodos , Trastuzumab/administração & dosagem , Trastuzumab/efeitos adversos , GencitabinaRESUMO
The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor ErbB-3/imunologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , HumanosRESUMO
The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Receptor ErbB-3/metabolismo , Animais , Biomarcadores Tumorais , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.
Assuntos
Anticorpos Monoclonais/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Especificidade de Anticorpos , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Epitopos/química , Epitopos/imunologia , Feminino , Proteína Forkhead Box O1 , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Receptor ErbB-2/química , Receptor ErbB-3/química , Receptor ErbB-3/imunologia , Trastuzumab , Carga Tumoral/efeitos dos fármacosRESUMO
Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients' treatment in oncology.