RESUMEN
Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.
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Receptores ErbB/inmunología , Exosomas/inmunología , Inmunidad Innata/inmunología , Neoplasias/inmunología , Virosis/inmunología , Adulto , Animales , Receptores ErbB/metabolismo , Exosomas/metabolismo , Femenino , Humanos , Huésped Inmunocomprometido/inmunología , MAP Quinasa Quinasa Quinasa 2/inmunología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana EdadRESUMEN
Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.
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Receptores ErbB/inmunología , Interleucina-13/inmunología , Interleucina-33/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células Th2/inmunología , Anfirregulina/inmunología , Anfirregulina/metabolismo , Animales , Línea Celular , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expresión Génica/genética , Expresión Génica/inmunología , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nematospiroides dubius/inmunología , Nematospiroides dubius/fisiología , Nocardia/inmunología , Nocardia/fisiología , Nocardiosis/inmunología , Nocardiosis/metabolismo , Nocardiosis/microbiología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/metabolismo , Infecciones por Strongylida/parasitología , Células Th2/metabolismoRESUMEN
Human IgA Abs engage neutrophils for cancer immunotherapy more effectively than IgG Abs. Previous studies demonstrated that engineering approaches improved biochemical and functional properties. In this study, we report a novel, to our knowledge, IgA2 Ab against the epidermal growth factor receptor generated by protein engineering and polymerization. The resulting molecule demonstrated a covalent linkage of L and H chains and an effective polymerization by the joining chain. The engineered dimer outperformed its monomeric variant in functional experiments on Fab-mediated modes of action and binding to the Fc receptor. The capacity to engage neutrophils for Ab-dependent cell-mediated cytotoxicity (ADCC) of adherent growing target cancer cells was cell line dependent. Although the engineered dimer displayed a long-term efficacy against the vulva carcinoma cell line A431, there was a notable in-efficacy against human papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC) cell lines. However, the highly engineered IgA Abs triggered a neutrophil-mediated cytotoxicity against HPV+ HNSCC cell lines. Short-term ADCC efficacy correlated with the target cells' epidermal growth factor receptor expression and the ability of cancer cell-conditioned media to enhance the CD147 surface level on neutrophils. Notably, the HPV+ HNSCC cell lines demonstrated a significant increment in releasing soluble CD147 and a reduced induction of membranous CD147 on neutrophils compared with HPV- cells. Although membranous CD147 on neutrophils may impair proper IgA-Fc receptor binding, soluble CD147 enhanced the IgA-neutrophil-mediated ADCC in a dose-dependent manner. Thus, engineering IgA Abs and impedance-based ADCC assays provided valuable information regarding the target-effector cell interaction and identified CD147 as a putative critical parameter for neutrophil-mediated cytotoxicity.
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Citotoxicidad Celular Dependiente de Anticuerpos , Basigina , Receptores ErbB , Neoplasias de Cabeza y Cuello , Inmunoglobulina A , Neutrófilos , Ingeniería de Proteínas , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Neutrófilos/inmunología , Receptores ErbB/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular Tumoral , Inmunoglobulina A/inmunología , Basigina/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapiaRESUMEN
The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.
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ADP Ribosa Transferasas , Receptores ErbB , Exotoxinas , Neoplasias de Cabeza y Cuello , Inmunoglobulina G , Inmunotoxinas , Exotoxina A de Pseudomonas aeruginosa , Factores de Virulencia , Humanos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Receptores ErbB/inmunología , Animales , Inmunotoxinas/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Ratones , Inmunoglobulina G/farmacología , Línea Celular Tumoral , Exotoxinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Cetuximab/farmacología , Ratones Desnudos , Toxinas Bacterianas , Apoptosis/efectos de los fármacos , Ratones Endogámicos BALB C , Femenino , Movimiento Celular/efectos de los fármacos , Antineoplásicos/farmacologíaRESUMEN
BACKGROUND: Antibody-drug conjugates have promising clinical activity in the treatment of solid tumours. BL-B01D1 is a first-in-class EGFR-HER3 bispecific antibody-drug conjugate. We aimed to assess the safety and preliminary antitumour activity of BL-B01D1 in patients with locally advanced or metastatic solid tumours. METHODS: This first-in-human, open-label, multicentre, dose-escalation and dose-expansion phase 1 trial was conducted in seven hospitals in China, enrolling patients aged 18-75 years (dose escalation; phase 1a) or older than 18 years (dose expansion; phase 1b), with a life expectancy of at least 3 months, an Eastern Cooperative Oncology Group performance status of 0-1, and histologically or cytologically confirmed locally advanced or metastatic solid tumours that had progressed on current standard treatment. In the phase 1a i3+3 design, patients received intravenous BL-B01D1 at three different schedules: 0·27 mg/kg, 1·5 mg/kg, and 3·0 mg/kg weekly; 2·5 mg/kg, 3·0 mg/kg, and 3·5 mg/kg on days 1 and 8 of each cycle every 3 weeks; or 5·0 mg/kg and 6·0 mg/kg on day 1 of each cycle every 3 weeks. The primary objectives of phase 1a were to identify the safety, maximum tolerated dose, and dose-limiting toxicity. In phase 1b, patients were treated in two schedules: 2·5 and 3·0 mg/kg on days 1 and 8 every 3 weeks, or 4·5, 5·0, and 6·0 mg/kg on day 1 every 3 weeks. The primary objectives of phase 1b were to assess the safety and recommended phase 2 dose of BL-B01D1, and objective response rate was a key secondary endpoint. Safety was analysed in all patients with safety records who received at least one dose of BL-B01D1. Antitumour activity was assessed in the activity analysis set which included all patients who received at least one dose of BL-B01D1 every 3 weeks. This trial is registered with China Drug Trials, CTR20212923, and ClinicalTrials.gov, NCT05194982, and recruitment is ongoing. FINDINGS: Between Dec 8, 2021, and March 13, 2023, 195 patients (133 [65%] men and 62 [32%] women; 25 in phase 1a and 170 in phase 1b) were consecutively enrolled, including 113 with non-small-cell lung cancer, 42 with nasopharyngeal carcinomas, 13 with small-cell lung cancer, 25 with head and neck squamous cell carcinoma, one with thymic squamous cell carcinoma, and one with submandibular lymphoepithelioma-like carcinoma. In phase 1a, four dose-limiting toxicities were observed (two at 3·0 mg/kg weekly and two at 3·5 mg/kg on days 1 and 8 every 3 weeks; all were febrile neutropenia), thus the maximum tolerated dose was reached at 3·0 mg/kg on days 1 and 8 every 3 weeks and 6·0 mg/kg on day 1 every 3 weeks. Grade 3 or worse treatment-related adverse events occurred in 139 (71%) of 195 patients; the most common of which were neutropenia (91 [47%]), anaemia (76 [39%]), leukopenia (76 [39%]), and thrombocytopenia (63 [32%]). 52 (27%) patients had a dose reduction and five (3%) patients discontinued treatment due to treatment-related adverse events. One patient was reported as having interstitial lung disease. Treatment-related deaths occurred in three (2%) patients (one due to pneumonia, one due to septic shock, and one due to myelosuppression). In 174 patients evaluated for activity, median follow-up was 6·9 months (IQR 4·5-8·9) and 60 (34%; 95% CI 27-42) patients had an objective response. INTERPRETATION: Our results suggest that BL-B01D1 has preliminary antitumour activity in extensively and heavily treated advanced solid tumours with an acceptable safety profile. Based on the safety and antitumour activity data from both phase 1a and 1b, 2·5 mg/kg on days 1 and 8 every 3 weeks was selected as the recommended phase 2 dose in Chinese patients. FUNDING: Sichuan Baili Pharmaceutical. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.
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Anticuerpos Biespecíficos , Receptores ErbB , Inmunoconjugados , Neoplasias , Receptor ErbB-3 , Humanos , Persona de Mediana Edad , Masculino , Femenino , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/uso terapéutico , Anciano , Adulto , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inmunoconjugados/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/inmunología , Adulto Joven , Dosis Máxima Tolerada , Adolescente , Metástasis de la Neoplasia , China , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
Our study aimed to expand tumor-infiltrating lymphocytes (TILs) from primary non-small cell lung cancers (NSCLCs) and evaluate their reactivity against tumor cells. We expanded TILs from 103 primary NSCLCs using histopathological analysis, flow cytometry, IFN-γ release assays, cell-mediated cytotoxicity assays, and in vivo efficacy tests. TIL expansion was observed in all cases, regardless of EGFR mutation status. There was also an increase in the median CD4+/CD8+ ratio during expansion. In post-rapid expansion protocol (REP) TILs, 13 out of 16 cases, including all three cases with EGFR mutations, exhibited a two-fold or greater increase in IFN-γ secretion. The cytotoxicity assay revealed enhanced tumor cell death in three of the seven cases, two of which had EGFR mutations. In vivo functional testing in a patient-derived xenograft model showed a reduction in tumor volume. The anti-tumor activity of post-REP TILs underscores their potential as a therapeutic option for advanced NSCLC, irrespective of mutation status.
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Carcinoma de Pulmón de Células no Pequeñas , Receptores ErbB , Neoplasias Pulmonares , Linfocitos Infiltrantes de Tumor , Mutación , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Receptores ErbB/genética , Receptores ErbB/inmunología , Animales , Femenino , Masculino , Persona de Mediana Edad , Anciano , Ratones , Interferón gamma/genética , Interferón gamma/inmunología , AdultoRESUMEN
Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands. In this study, we have investigated the potential for protein-based octomeric chemically self-assembled nanorings (CSANs) to be used for anticancer drug delivery. The CSANs are composed of a DHFR-DHFR fusion protein incorporating an EGFR-targeting fibronectin and the anticancer drug MMAE conjugated through a C-terminal farnesyl azide. The anti-EGFR-MMAE CSANs were shown to undergo rapid internalization and have potent cytotoxicity to cancer cells across a 9000-fold difference in EGFR expression. In addition, anti-EGFR-MMAE CSANs were shown to induce immunological cell death. Thus, multivalent and modular CSANs are a potential alternative anticancer drug delivery platform with the capability of targeting tumor cells with heterogeneous antigen expression while activating the anticancer immune response.
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Antineoplásicos , Sistemas de Liberación de Medicamentos , Muerte Celular Inmunogénica , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Receptores ErbB/metabolismo , Receptores ErbB/inmunología , Muerte Celular Inmunogénica/efectos de los fármacos , Nanopartículas/química , Nanoestructuras/químicaRESUMEN
LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.
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Receptores ErbB , Macaca fascicularis , Animales , Receptores ErbB/inmunología , Humanos , Masculino , Femenino , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Biespecíficos/efectos adversos , Relación Dosis-Respuesta a DrogaRESUMEN
Type 2 inflammatory responses can be elicited by diverse stimuli, including toxins, venoms, allergens, and infectious agents, and play critical roles in resistance and tolerance associated with infection, wound healing, tissue repair, and tumor development. Emerging data suggest that in addition to characteristic type 2-associated cytokines, the epidermal growth factor (EGF)-like molecule Amphiregulin (AREG) might be a critical component of type 2-mediated resistance and tolerance. Notably, numerous studies demonstrate that in addition to the established role of epithelial- and mesenchymal-derived AREG, multiple leukocyte populations including mast cells, basophils, group 2 innate lymphoid cells (ILC2s), and a subset of tissue-resident regulatory CD4(+) T cells can express AREG. In this review, we discuss recent advances in our understanding of the AREG-EGF receptor pathway and its involvement in infection and inflammation and propose a model for the function of this pathway in the context of resistance and tissue tolerance.
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Familia de Proteínas EGF/inmunología , Receptores ErbB/inmunología , Tolerancia Inmunológica/inmunología , Inflamación/inmunología , Cicatrización de Heridas/inmunología , Anfirregulina , Animales , Helmintiasis/inmunología , Humanos , Gripe Humana/inmunología , Ratones , Neoplasias/inmunología , Infecciones por Orthomyxoviridae/inmunología , Regeneración , Escape del Tumor/inmunologíaRESUMEN
Staphylococcus aureus skin colonization is universal in atopic dermatitis and common in cancer patients treated with epidermal growth factor receptor inhibitors. However, the causal relationship of dysbiosis and eczema has yet to be clarified. Herein, we demonstrate that Adam17(fl/fl)Sox9-(Cre) mice, generated to model ADAM17-deficiency in human, developed eczematous dermatitis with naturally occurring dysbiosis, similar to that observed in atopic dermatitis. Corynebacterium mastitidis, S. aureus, and Corynebacterium bovis sequentially emerged during the onset of eczematous dermatitis, and antibiotics specific for these bacterial species almost completely reversed dysbiosis and eliminated skin inflammation. Whereas S. aureus prominently drove eczema formation, C. bovis induced robust T helper 2 cell responses. Langerhans cells were required for eliciting immune responses against S. aureus inoculation. These results characterize differential contributions of dysbiotic flora during eczema formation, and highlight the microbiota-host immunity axis as a possible target for future therapeutics in eczematous dermatitis.
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Dermatitis Atópica/inmunología , Disbiosis/inmunología , Eccema/inmunología , Células de Langerhans/inmunología , Piel/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Proteína ADAM17 , Animales , Antibacterianos/farmacología , Corynebacterium/inmunología , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/genética , Dermatitis Atópica/microbiología , Disbiosis/tratamiento farmacológico , Disbiosis/genética , Disbiosis/microbiología , Eccema/tratamiento farmacológico , Eccema/genética , Eccema/microbiología , Receptores ErbB/genética , Receptores ErbB/inmunología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Integrasas/genética , Integrasas/inmunología , Células de Langerhans/efectos de los fármacos , Células de Langerhans/microbiología , Células de Langerhans/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/inmunología , Transducción de Señal , Piel/efectos de los fármacos , Piel/microbiología , Piel/patología , Staphylococcus aureus/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/microbiología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.
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Antígenos B7/metabolismo , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Neoplasias/inmunología , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/metabolismo , Antígenos B7/genética , Línea Celular Tumoral , Cetuximab/genética , Citocinas/metabolismo , Citotoxicidad Inmunológica , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Ingeniería Genética , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Mediadores de Inflamación/metabolismo , Células Asesinas Naturales/trasplante , Activación de Linfocitos , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Neoplasias/terapia , Unión Proteica , Transducción de SeñalRESUMEN
Necitumumab enhances antitumor immunity by decreasing the PD-L1 expression; it is expected to improve the prognosis of patients treated with an immune checkpoint inhibitor(ICI)by inhibiting the IL-8 expression. Since the combined effect of necitumumab and PD-L1 inhibitor was confirmed in an in vivo study conducted in transgenic mice, further antitumor effects can be expected by the combined use of necitumumab and ICI.
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Anticuerpos Monoclonales Humanizados , Inhibidores de Puntos de Control Inmunológico , Animales , Humanos , Ratones , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Proyectos de Investigación , Receptores ErbB/inmunologíaRESUMEN
A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non-small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF ß-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.
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Anticuerpos Biespecíficos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Descubrimiento de Drogas , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Proteínas Proto-Oncogénicas c-met/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed hybrid cancer therapy that directly kills cancer cells as well as producing a therapeutic host immune response. Conventional immunotherapies, such as immune-activating cytokine therapy, checkpoint inhibition, engineered T cells and suppressor cell depletion, do not directly destroy cancer cells, but rely exclusively on activating the immune system. NIR-PIT selectively destroys cancer cells, leading to immunogenic cell death that initiates local immune reactions to released cancer antigens from dying cancer cells. These are characterized by rapid maturation of dendritic cells and priming of multi-clonal cancer-specific cytotoxic T cells that kill cells that escaped the initial direct effects of NIR-PIT. The NIR-PIT can be applied to a wide variety of cancers either as monotherapy or in combination with conventional immune therapies to further activate anti-cancer immunity. A global Phase 3 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03769506) of NIR-PIT targeting the epidermal growth factor receptor (EGFR) in patients with recurrent head and neck cancer is underway, employing RM1929/ASP1929, a conjugate of anti-EGFR antibody (cetuximab) plus the photo-absorber IRDye700DX (IR700). NIR-PIT has been given fast-track recognition by regulators in the USA and Japan. A variety of imaging methods, including direct IR700 fluorescence imaging, can be used to monitor NIR-PIT. As experience with NIR-PIT grows, additional antibodies will be employed to target additional antigens on other cancers or to target immune-suppressor cells to enhance host immunity. NIR-PIT will be particularly important in patients with localized and locally advanced cancers and may help such patients avoid side-effects associated with surgery, radiation and chemotherapy.
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Neoplasias de Cabeza y Cuello/terapia , Inmunoterapia/métodos , Rayos Infrarrojos/uso terapéutico , Linfocitos T Citotóxicos/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Cetuximab/uso terapéutico , Terapia Combinada , Células Dendríticas/inmunología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Indoles/uso terapéutico , Activación de Linfocitos/inmunología , Compuestos de Organosilicio/uso terapéuticoRESUMEN
Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients.
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Receptores ErbB/inmunología , Glicoproteínas/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Linfocitos T Reguladores/inmunología , Anfirregulina , Animales , Anticuerpos Neutralizantes/farmacología , Comunicación Celular/inmunología , Colitis/inducido químicamente , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Familia de Proteínas EGF , Receptores ErbB/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Glicoproteínas/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Activación de Linfocitos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
Glioblastoma (GBM) is the deadliest brain malignancy without effective treatments. Here, we reported that epidermal growth factor receptor-targeted chimeric antigen receptor T cells (EGFR CAR-T) were effective in suppressing the growth of GBM cells in vitro and xenografts derived from GBM cell lines and patients in mice. However, mice soon acquired resistance to EGFR CAR-T cell treatment, limiting its potential use in the clinic. To find ways to improve the efficacy of EGFR CAR-T cells, we performed genomics and transcriptomics analysis for GBM cells incubated with EGFR CAR-T cells and found that a large cohort of genes, including immunosuppressive genes, as well as enhancers in vicinity are activated. BRD4, an epigenetic modulator functioning on both promoters and enhancers, was required for the activation of these immunosuppressive genes. Accordingly, inhibition of BRD4 by JQ1 blocked the activation of these immunosuppressive genes. Combination therapy with EGFR CAR-T cells and JQ1 suppressed the growth and metastasis of GBM cells and prolonged survival in mice. We demonstrated that transcriptional modulation by targeting epigenetic regulators could improve the efficacy of immunotherapy including CAR-T, providing a therapeutic avenue for treating GBM in the clinic.
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Azepinas/administración & dosificación , Neoplasias Encefálicas/terapia , Proteínas de Ciclo Celular/metabolismo , Receptores ErbB/inmunología , Glioblastoma/terapia , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Factores de Transcripción/metabolismo , Triazoles/administración & dosificación , Animales , Azepinas/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Epigénesis Genética/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ratones , Metástasis de la Neoplasia , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3ß oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.
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Adenocarcinoma/tratamiento farmacológico , Antígeno Ca-125/genética , Carcinogénesis/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Receptores ErbB/genética , Proteínas de la Membrana/genética , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales/farmacología , Antígeno Ca-125/inmunología , Carcinogénesis/inmunología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Ratones , Metástasis de la Neoplasia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Transducción de SeñalRESUMEN
The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50 values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.
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Proteínas Anfibias/farmacología , Antineoplásicos Inmunológicos/farmacología , Inmunotoxinas/farmacología , Ingeniería de Proteínas , Ribonucleasas/farmacología , Anticuerpos de Cadena Única/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Antineoplásicos Inmunológicos/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión de Anticuerpos , Células CHO , Línea Celular Tumoral , Cricetulus , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Inmunotoxinas/genética , Inmunotoxinas/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Rana pipiens , Ribonucleasas/genética , Ribonucleasas/metabolismo , Anticuerpos de Cadena Única/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.
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Receptores ErbB/inmunología , Neoplasias de la Vesícula Biliar/inmunología , Huésped Inmunocomprometido/fisiología , Midkina/efectos adversos , Proliferación Celular/genética , China/epidemiología , Receptores ErbB/antagonistas & inhibidores , Neoplasias de la Vesícula Biliar/epidemiología , Neoplasias de la Vesícula Biliar/fisiopatología , Humanos , Midkina/genética , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/estadística & datos numéricos , Transducción de Señal/genética , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/estadística & datos numéricos , Secuenciación del Exoma/métodos , Secuenciación del Exoma/estadística & datos numéricosRESUMEN
The epidermal growth factor receptor extracellular domain III (EGFR-ECDIII) protein is a promising target of anti-cancer research, and its production in Escherichia coli would thus represent significant benefits. However, despite its moderate size (19 kDa), the expression of EGFR-ECDIII in E.coli is hampered by the presence of multiple cysteines producing misfolded proteins with incorrect S-S bonds. In our study, we show that a short 12-residue solubility enhancing peptide (SEP) tag containing nine arginines (C9R) attached at the C-terminus of EGFR-ECDIII reduces the inclusion body formation and increases the final yield by six times (20 mg/L). EGFR-ECDIII-C9R purified from the soluble fraction eluted as a sharp single RP-HPLC peak, suggesting a single S-S bond pairing. Biophysical characterization using circular dichroism, fluorescence, and light scattering confirmed its native-like properties together with reversible thermal denaturation. The binding activity of EGFR-ECDIII-C9R to anti-EGFR-VHH7D12, a single-domain antibody with specific binding to the ECDIII, was assessed by sandwich ELISA. Further, we produced anti-EGFR-ECDIII-C9R antisera in mouse models and anti-sera inhibited A431 cancer cells' growth. These results demonstrate that the SEP tag enables the rapid production of the multiple disulfide-bonded EGFR-ECDIII in E. coli having native-like biophysical properties and producing neutralizing antibodies.