RESUMEN
UNLABELLED: To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1ß, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCE: Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.
Asunto(s)
Células Dendríticas/inmunología , Factores Inmunológicos/inmunología , Neoplasias/terapia , ARN Bicatenario/inmunología , ARN Viral/inmunología , Saccharomyces cerevisiae/virología , Receptor Toll-Like 3/inmunología , Animales , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , ARN Bicatenario/aislamiento & purificación , ARN Bicatenario/uso terapéutico , ARN Viral/aislamiento & purificación , ARN Viral/uso terapéutico , Análisis de SupervivenciaRESUMEN
Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.
Asunto(s)
Aminas/química , Derivados del Benceno/química , Reactivos de Enlaces Cruzados/química , Inmunoconjugados/química , Compuestos de Sulfhidrilo/química , Línea Celular , Halogenación , Humanos , Inmunoconjugados/sangre , Maleimidas/químicaRESUMEN
Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.
Asunto(s)
Alquinos/química , Maleimidas/química , Compuestos de Sulfhidrilo/química , Línea Celular Tumoral , Humanos , Inmunoconjugados/química , Indicadores y Reactivos/química , CinéticaRESUMEN
BACKGROUND: We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643). METHODS: Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced. RESULTS: DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8⺠and CD4⺠T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a⺠CD8⺠T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model. CONCLUSIONS: We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development.
Asunto(s)
Formación de Anticuerpos , Hepatitis C/prevención & control , Vacunación/métodos , Vacunas contra Hepatitis Viral/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Genotipo , Hepacivirus , Hepatitis C/inmunología , Inmunización Secundaria , Interferón gamma/sangre , Interleucina-2/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Necrosis Tumoral alfa/sangre , Vacunas de ADN/inmunología , Vacunas contra Hepatitis Viral/genéticaRESUMEN
BACKGROUND: Primary cutaneous lymphomas (CLs), characterized by an accumulation of clonal T or B lymphocytes preferentially localized in the skin, have been successfully treated with interferons (IFNs) which counterbalance the Th2-immunosuppressive state associated with this pathology. In a phase I/II clinical trial, we correlated the local immune infiltrate and the anti-tumor effects of repeated intralesional administrations of an adenovirus vector expressing human interferon-gamma (IFN-g) termed TG1042, in patients with advanced primary cutaneous T-cell lymphomas (CTCL) or multilesional cutaneous B-cell lymphomas (CBCL). METHODS: For each patient, variation in time of specific lymphocyte populations, defined by immunohistochemical stainings, was assessed in biopsies of injected lesions. For each patient, the change in local immune response was associated with the patient's objective response at the end of the study. RESULTS: Immunohistochemical analyses of biopsies indicate that infiltration of CD8+ T lymphocytes and of TIA-1+ cytotoxic T-cells in lesions injected with TG1042 correlates with clinical benefit. CONCLUSIONS: These data suggest for the first time that a CD8+ cytotoxic infiltrate, induced by local expression of IFN-g correlates with a clinical response. TRIAL REGISTRATION: The phase I step (TG1042.01) does not have a registration number. The phase II step (TG1042.06) registration number was NCT00394693.
Asunto(s)
Vectores Genéticos/administración & dosificación , Linfocitos Infiltrantes de Tumor/inmunología , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/terapia , Microambiente Tumoral/inmunología , Progresión de la Enfermedad , Humanos , Linfoma Cutáneo de Células T/patología , Resultado del TratamientoRESUMEN
BACKGROUND & AIMS: Therapy for chronic hepatitis C (CHC) has limited efficacy, adverse effects, and high costs. Cohort and vaccine-based preclinical studies have indicated the importance of T-cell-based immunity in controlling viral infection. TG4040 is a recombinant poxvirus vaccine that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. We performed a phase I clinical trial to assess the safety, immunogenicity, and early antiviral efficacy of TG4040 in patients with CHC. METHODS: In an open-label, dose-escalating study, patients with mild CHC (genotype 1) were assigned to 3 groups of 3 patients each; they received subcutaneous injections of 106, 107, or 108 plaque-forming units of TG4040 on study days 1, 8, and 15. Six additional patients were given the highest dose of vaccine (108 plaque-forming units). Patients were followed for 6 months after the last injection. T-cell-based and antibody responses and levels of HCV RNA were measured. RESULTS: All 3 doses of TG4040 were well tolerated, without serious adverse events. Vaccine-induced HCV-specific cellular immune responses were observed in 5 of the 15 patients (33%). A transient decrease in circulating levels of HCV RNA, from -0.52 log10 to -1.24 log10, was observed in 8 patients; in 5 patients, the lowest level of HCV RNA was observed on day 37, after the first injection. The most pronounced decrease in viral load occurred in 2 patients, who also had marked vaccine-induced T-cell responses. CONCLUSIONS: In patients with CHC, the viral-vector-based vaccine TG4040 had a good safety profile, induced HCV-specific cellular immune responses, and reduced viral load. This vaccine should be investigated in further clinical studies, in combination with standard of care.
Asunto(s)
Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Poxviridae/inmunología , Linfocitos T/efectos de los fármacos , Carga Viral/efectos de los fármacos , Vacunas Virales/farmacología , Adulto , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/efectos adversos , Vacunas Virales/uso terapéuticoRESUMEN
BACKGROUND: Chemotherapy is the standard of care for advanced stages of non-small-cell lung cancer (NSCLC). TG4010 is a targeted immunotherapy based on a poxvirus (modified vaccinia virus Ankara) that codes for MUC1 tumour-associated antigen and interleukin 2. This study assessed TG4010 in combination with first-line chemotherapy in advanced NSCLC. METHODS: 148 patients with advanced (stage IIIB [wet] or IV) NSCLC expressing MUC1 by immunohistochemistry, and with performance status 0 or 1, were enrolled in parallel groups in this open-label, phase 2B study. 74 patients were allocated to the combination therapy group, and received TG4010 (10(8) plaque forming units) plus cisplatin (75 mg/m(2) on day 1) and gemcitabine (1250 mg/m(2) on days 1 and 8) repeated every 3 weeks for up to six cycles. 74 patients allocated to the control group received the same chemotherapy alone. Patients were allocated using a dynamic minimisation procedure stratified by centre, performance status, and disease stage. The primary endpoint was 6-month progression-free survival (PFS), with a target rate of 40% or higher in the experimental group. Analyses were done on an intention-to-treat basis. This study is completed and is registered with ClinicalTrials.gov, number NCT00415818. FINDINGS: 6-month PFS was 43·2% (32 of 74; 95% CI 33·4-53·5) in the TG4010 plus chemotherapy group, and 35·1% (26 of 74; 25·9-45·3) in the chemotherapy alone group. Fever, abdominal pain, and injection-site pain of any grade according to National Cancer Institute Common Toxicity Criteria were more common in the TG4010 group than in the chemotherapy alone group: 17 of 73 patients (23·3%) versus six of 72 (8·3%), 12 (16·4%) versus two (2·8%), and four (5·5%) versus zero (0%), respectively. The most common grade 3-4 adverse events were neutropenia (33 [45·2%] of patients in the TG4010 plus chemotherapy group vs 31 [43·1%] in the chemotherapy alone group) and fatigue (18 [24·7%] vs 13 [18·1%]); the only grade 3-4 events that differed significantly between groups were anorexia (three [4·1%] vs 10 [13·9%]) and pleural effusion (none vs four [5·6%]). 38 of 73 patients (52·1%) in the TG4010 plus chemotherapy group and 34 of 72 (47·2%) in the chemotherapy alone group had at least one serious adverse event. INTERPRETATION: This phase 2B study suggests that TG4010 enhances the effect of chemotherapy in advanced NSCLC. A confirmatory phase 2B-3 trial has been initiated. FUNDING: Transgene SA, Advanced Diagnostics for New Therapeutic Approaches (ADNA)/OSEO.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/tratamiento farmacológico , Glicoproteínas de Membrana/uso terapéutico , Adulto , Anciano , Antimetabolitos Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Vacunas contra el Cáncer/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Quimioterapia Adyuvante , Distribución de Chi-Cuadrado , Cisplatino/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Supervivencia sin Enfermedad , Europa (Continente) , Femenino , Humanos , Interleucina-2/genética , Interleucina-2/inmunología , Estimación de Kaplan-Meier , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Glicoproteínas de Membrana/efectos adversos , Persona de Mediana Edad , Mucina-1/genética , Mucina-1/inmunología , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Vacunas Sintéticas , Virus Vaccinia/genética , GemcitabinaRESUMEN
Immunotherapy of cancer has long been considered as an attractive therapeutic approach but with no impact on clinical practice. Two clinical protocols of immunotherapy, one based on a cancer vaccine in patients with prostate cancer or melanoma and the other using an immunomodulator targeting T cells (anti-CTLA4 mAb) in melanoma patients, have demonstrated clinical efficacy in two phase III clinical trials. To improve these encouraging clinical results, biomarkers to better select patients which may benefit from this therapy are actively searched. In addition, immunosuppression associated with cancer has to be overcome to allow a better immunostimulation. In contrast to chemotherapy, clinical variables to monitor the efficacy of immunotherapy has to be revisited and overall survival appears to be a better endpoint than clinical response defined by the RECIST criteria. Combination of immunotherapy with conventional treatments (chemotherapy, anti-angiogenic, etc.) should further improve this approach both in its effectiveness and in its clinical indications.
Asunto(s)
Inmunoterapia/tendencias , Neoplasias/terapia , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias/inmunología , Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Ensayos Clínicos como Asunto , Terapia Combinada , Predicción , Humanos , Inmunidad Celular , Inmunoterapia Activa , Ratones , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Lesiones Precancerosas/terapia , Análisis de Supervivencia , Subgrupos de Linfocitos T/inmunología , Terapias en InvestigaciónRESUMEN
Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Nevertheless, it is now recognized that the same vaccination strategies used for prophylactic vaccinations against infectious diseases, cannot necessarily be used for therapeutic cancer vaccination. Cancer patients are usually immunosuppressed and most cancer-associated antigens are 'self-antigens', making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulation techniques are under investigation in an effort to bolster patients' immune systems and to overcome immune tolerance to self antigens. Strategies to stimulate antigen presentation, T-cell reactivity and innate immune activity are under investigation. Several clinical trials have been conducted to evaluate therapeutic cancer vaccines in patients, and attractive protocols, including those that combine the stimulation of specific T-cells and chemotherapy, or strategies to block immune regulation, are beginning to show some success. This feature review considers strategies for the development of effective therapeutic cancer vaccines, and highlights select vaccines that have already entered the clinic.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas contra el Cáncer/uso terapéutico , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/efectos de los fármacos , Vías de Administración de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias/inmunología , Neoplasias/prevención & control , Vacunación/métodos , Vacunación/tendenciasRESUMEN
We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.
Asunto(s)
Linfocitos B/inmunología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina E/inmunología , Cadenas epsilon de Inmunoglobulina/inmunología , Linfocitos B/citología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Células Cultivadas , Técnicas de Cultivo , ADN Circular/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Sangre Fetal/citología , Humanos , Inmunoglobulina E/genética , Cadenas epsilon de Inmunoglobulina/genética , Separación Inmunomagnética/métodos , Activación de Linfocitos , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3beta (MIP-3beta) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Células de Langerhans/metabolismo , Antígenos CD/biosíntesis , Antígenos CD1/biosíntesis , Antígeno B7-2 , Proteínas de la Membrana Bacteriana Externa/farmacología , División Celular/inmunología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Antígenos HLA-DR/biosíntesis , Humanos , Inmunofenotipificación , Células de Langerhans/citología , Células de Langerhans/inmunología , Glicoproteínas de Membrana/biosíntesis , Unión Proteica , Linfocitos T/citología , Linfocitos T/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⺠T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Portadores de Fármacos , Inmunidad Celular , Proteínas Oncogénicas Virales/inmunología , Vacunas contra Papillomavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/genética , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales/genética , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Interleucinas/inmunología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Humanos , Región Variable de Inmunoglobulina/química , Factor Estimulante de Colonias de Macrófagos/inmunología , Ratones , Modelos Químicos , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Alineación de SecuenciaRESUMEN
OBJECTIVE: Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA. METHODS: CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis. RESULTS: CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1- monocytes. CONCLUSION: CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Artritis Experimental/patología , Artritis Reumatoide/patología , Huesos/patología , Cartílago/patología , Osteoartritis/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Estudios de Casos y Controles , Células Dendríticas/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Monocitos/metabolismo , Monocitos/patología , Osteoartritis/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Therapeutic vaccines are different from the well-known prophylactic vaccines in that they are designed to treat patients already suffering from a disease instead of preventing the disease in healthy individuals. Several therapeutic vaccines are today in late-stage clinical development for non-small-cell lung cancer. These vaccines use different approaches including peptides, cell lines and viral vectors, and explore different settings within the pathology. Some are given in monotherapy while others are combined with the classic therapies used with non-small-cell lung cancer. This review gives a summary of the therapeutic vaccines currently in late-stage clinical development for non-small-cell lung cancer.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/terapia , Inmunoterapia Activa/métodos , Neoplasias Pulmonares/terapia , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Ensayos Clínicos como Asunto , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/uso terapéuticoRESUMEN
Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+) CD163(+) M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+)CD163(+) M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Macrófagos/inmunología , Neoplasias Experimentales/terapia , Osteoclastos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Análisis de SupervivenciaRESUMEN
Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Osteólisis/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Interleucina-6/inmunología , Interleucina-6/metabolismo , Factor Estimulante de Colonias de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Monocitos/metabolismo , Células 3T3 NIH , Osteoclastos/efectos de los fármacos , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteólisis/inmunología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
Cancer immunotherapy is hampered by the immunosuppression maintained by regulatory T cells (Tregs) in tumor-bearing hosts. Stimulation of the Toll-like receptor 2 (TLR2) by Pam3Cys is known to affect Treg-mediated suppression. We found that Pam3Cys increases the proliferation of both CD4(+) effector T cells (Teffs) and Tregs co-cultured in vitro, but did not induce the proliferation of Tregs alone upon CD3 and CD28 stimulation. In a mouse model of RMA-MUC1 tumors, Pam3Cys was administered either alone or in combination with a modified vaccinia ankara (MVA)-based mucin 1 (MUC1) therapeutic vaccine. The combination of Pam3Cys with MVA-MUC1 (1) diminished splenic Treg/CD4(+) T-cell ratios to those found in tumor-free mice, (2) stimulated a specific anti-MUC1 interferon γ (IFNγ) response and (3) had a significant therapeutic effect on tumor growth and mouse survival. When CD4(+) Teffs and Tregs were isolated from Pam3Cys-treated mice, Teffs had become resistant to Treg-mediated suppression while upregulating the expression of BclL-x(L). Tregs from Pam3Cys-treated mice were fully suppressive for Teffs from naïve mice. Bcl-x(L) was induced by Pam3Cys with different kinetics in Tregs and Teffs. Teff from Pam3Cys-treated mice produced increased levels of Th1 and Th2-type cytokines and an interleukin (IL)-6-dependent secretion of IL-17 was observed in Teff:Treg co-cultures, suggesting that TLR2 stimulation had skewed the immune response toward a Th17 profile. Our results show for the first time that in a tumor-bearing host, TLR2 stimulation with Pam3Cys affects both Tregs and Teffs, protects Teff from Treg-mediated suppression and has strong therapeutic effects when combined with an MVA-based antitumor vaccine.
RESUMEN
Tumor-associated antigens (TAAs) have been formulated into vaccines that, combined with adjuvants, cytokines or other strategies to boost the immune response, are now in clinical development. Both humoral and cellular immune responses to TAAs have been generated using these vaccines. This approach relies on the patient's own immune system generating an effective anti-tumor immune response. The advantage of this over therapy with monoclonal antibodies for the treatment of cancer is that multiple antigenic epitopes can be involved and the immune system is able to adapt to the most effective antigenic specificity for tumor growth control and rejection. In this article, we describe the clinical use of vaccinia virus, in particular modified vaccinia virus Ankara (MVA), to express TAAs in vivo and to stimulate an effective immune response to the cancer antigens.