RESUMEN
The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (KD) was 2.32 × 10-7 M and 2.83 × 10-9 M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity KD between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10-4 M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals.
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Receptores de HFE , Animales , Perros , Gatos , Masculino , Receptores de HFE/metabolismo , Receptores de HFE/genética , Receptores de HFE/inmunología , Anticoncepción/veterinaria , Anticoncepción/métodos , Células de Sertoli/metabolismoRESUMEN
Hyperandrogenemia and ovulatory dysfunction are hallmarks of polycystic ovary syndrome (PCOS), pointing to a deranged hypothalamus-pituitary-ovarian (HPO) axis. An autoimmune etiology of PCOS is suspected in a subset of patients due to the relatively high concordance of PCOS with common autoimmune diseases. For this reason, we tested the hypothesis that natural autoantibodies (aAb) to the follicle-stimulating hormone receptor (FSHR) or luteinizing hormone receptor (LHR) are prevalent in PCOS. To this end, new luminometric assays for quantifying aAb to the FSHR (FSHR-aAb) or LHR (LHR-aAb) were developed using full-length recombinant human receptors as fusion proteins with luciferase as reporter. Prevalence of FSHR-aAb and LHR-aAb was determined in serum samples from healthy controls and PCOS patients. Steroid hormone profiles were compared between patients with and without FSHR-aAb or LHR-aAb. Signal linearity and detection ranges were characterized and both methods passed basic performance quality checks. The analysis revealed a relatively low prevalence, with 4 out of 430 samples positive for FSHR-aAb in the control versus 11 out of 550 samples in the PCOS group, i.e., 0.9% versus 2.0%, respectively. Similarly, there were only 5 samples positive for LHR-aAb in the control versus 2 samples in the PCOS group, i.e., 1.2% versus 0.4%, respectively. Samples positive for FSHR-aAb displayed steroid hormones in the typical range of PCOS patients, whereas the two samples positive for LHR-aAb showed relatively elevated free testosterone in relation to total testosterone concentrations with unclear significance. We conclude that the FSHR and LHR constitute potential autoantigens in human subjects. However, the prevalence of specific autoantibodies to these receptors is relatively low, both in control subjects and in women with PCOS. It is therefore unlikely that autoimmunity to the LHR or FSHR constitutes a frequent cause of hyperandrogenemia or ovulatory dysfunction in PCOS.
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Autoanticuerpos/sangre , Síndrome del Ovario Poliquístico/inmunología , Receptores de HFE/inmunología , Receptores de HL/inmunología , Estudios de Casos y Controles , Femenino , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Síndrome del Ovario Poliquístico/sangre , Prevalencia , Receptores de HFE/genética , Receptores de HL/genética , Proteínas Recombinantes/inmunología , TestosteronaRESUMEN
Background: rs13405728 was identified as one of the most prevalent susceptibility loci for polycystic ovary syndrome (PCOS) in Han Chinese and Caucasian women. However, the target genes and potential mechanisms of the rs13405728 locus remain to be determined. Methods: Three-dimensional (3D) genome interactions from the ovary tissue were characterized via high-through chromosome conformation capture (Hi-C) and Capture Hi-C technologies to identify putative targets at the rs13405728 locus. Combined analyses of eQTL, RNA-Seq, DNase-Seq, ChIP-Seq, and sing-cell sequencing were performed to explore the molecular roles of these target genes in PCOS. PCOS-like mice were applied to verify the expression patterns. Results: Generally, STON1 and FSHR were identified as potential targets of the rs13405728 locus in 3D genomic interactions with epigenomic regulatory peaks, with STON1 (P=0.0423) and FSHR (P=0.0013) being highly expressed in PCOS patients. STON1 co-expressed genes were associated with metabolic processes (P=0.0008) in adipocytes (P=0.0001), which was validated in the fat tissue (P<0.0001) and ovary (P=0.0035) from fat-diet mice. The immune system process (GO:0002376) was enriched in FSHR co-expressed genes (P=0.0002) and PCOS patients (P=0.0002), with CD4 high expression in PCOS patients (P=0.0316) and PCOS-like models (P=0.0079). Meanwhile, FSHR expression was positively correlated with CD4 expression in PCOS patients (P=0.0252) and PCOS-like models (P=0.0178). Furthermore, androgen receptor (AR) was identified as the common transcription factor for STON1 and FSHR and positively correlated with the expression of STON1 (P=0.039) and FSHR (P=4e-06) in ovary tissues and PCOS-like mice. Conclusion: Overall, we identified STON1 and FSHR as potential targets for the rs13405728 locus and their roles in the processes of adipocyte metabolism and CD4 immune expression in PCOS, which provides 3D genomic insight into the pathogenesis of PCOS.
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Proteínas de la Membrana/genética , Síndrome del Ovario Poliquístico/genética , Receptores de HFE/genética , Factores Generales de Transcripción/genética , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Antígenos CD4/inmunología , Femenino , Expresión Génica , Sitios Genéticos , Genoma , Humanos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ovario/metabolismo , Síndrome del Ovario Poliquístico/inmunología , Síndrome del Ovario Poliquístico/metabolismo , Receptores Androgénicos/genética , Receptores de HFE/inmunología , Factores Generales de Transcripción/metabolismoRESUMEN
Ovarian cancer presents in 80% of patients as a metastatic disease, which confers it with dismal prognosis despite surgery and chemotherapy. However, it is an immunogenic disease, and the presence of intratumoral T cells is a major prognostic factor for survival. We used a synthetic consensus (SynCon) approach to generate a novel DNA vaccine that breaks immune tolerance to follicle-stimulating hormone receptor (FSHR), present in 50% of ovarian cancers but confined to the ovary in healthy tissues. SynCon FSHR DNA vaccine generated robust CD8+ and CD4+ cellular immune responses and FSHR-redirected antibodies. The SynCon FSHR DNA vaccine delayed the progression of a highly aggressive ovarian cancer model with peritoneal carcinomatosis in immunocompetent mice, and it increased the infiltration of anti-tumor CD8+ T cells in the tumor microenvironment. Anti-tumor activity of this FSHR vaccine was confirmed in a syngeneic murine FSHR-expressing prostate cancer model. Furthermore, adoptive transfer of vaccine-primed CD8+ T cells after ex vivo expansion delayed ovarian cancer progression. In conclusion, the SynCon FSHR vaccine was able to break immune tolerance and elicit an effective anti-tumor response associated with an increase in tumor-infiltrating T cells. FSHR DNA vaccination could help current ovarian cancer therapy after first-line treatment of FSHR+ tumors to prevent tumor recurrence.
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Vacunas contra el Cáncer/uso terapéutico , Neoplasias Ováricas/prevención & control , Receptores de HFE/inmunología , Vacunas de ADN/uso terapéutico , Animales , Vacunas contra el Cáncer/inmunología , Femenino , Citometría de Flujo , Células HEK293 , Humanos , Immunoblotting , Inmunoterapia/métodos , Ratones , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Vacunas de ADN/inmunologíaRESUMEN
In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.
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Anticuerpos , Células de la Granulosa/metabolismo , Receptores de HFE/inmunología , Animales , Bovinos , FemeninoRESUMEN
Improved molecular understanding of tumor microenvironment has resulted in the identification of various cancer cell targets for diagnostic and therapeutic interventions, including the receptor for the FSH, a glycoprotein hormone responsible for growth, maturation, and function of human reproductive system. The expression and localization of the FSH receptor (FSHR)-protein were associated with the tumor epithelial cells and/or with the peripheral tumor blood vessels. The available evidence indicates that in ovarian cancer, prostate cancer, and breast cancer, the tumor epithelial FSHR promotes proliferation, migration, and invasion of cancer cells. The vascular endothelial FSHR, detected in 11 types of solid tumors and 11 types of sarcomas, is involved in receptor-mediated transendothelial transport of FSH, tumor angiogenesis, and vascular remodeling. In contrast to intratumor vessels, which are abnormal and disorganized, the FSHR-positive blood microvessels are arranged in a hierarchical pattern: arterioles-capillaries-venules. The FSHR-positive blood vessels make connections between the intratumor vessels and the general blood circulation of patients. In this mini-review, I summarize these studies and discuss the rationale for developing a strategy for cancer therapy based on FSHR expressed on the luminal endothelial cell surface of blood vessels located in the peritumoral area rather than endothelial markers expressed in the core of tumors. Because FSHR is a common marker of peritumoral vessels, therapeutic agents coupled to anti-FSHR humanized antibodies should in principle be applicable to a wide range of tumor types.
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Antineoplásicos/administración & dosificación , Endotelio Vascular/metabolismo , Neoplasias/tratamiento farmacológico , Receptores de HFE/metabolismo , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Masculino , Terapia Molecular Dirigida , Invasividad Neoplásica , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores de HFE/inmunología , Sarcoma/irrigación sanguínea , Sarcoma/tratamiento farmacológico , Sarcoma/metabolismo , Sarcoma/patología , Microambiente TumoralRESUMEN
PURPOSE: In recent studies, follicle-stimulating hormone receptors (FSHRs) have been reported in a wide range of malignant and benign tumours, depending on the type of antibody used. Using two commercially available antibodies (monoclonal and polyclonal), the current research attempted to demonstrate the usefulness of each antibody for investigating FSHRs in non-canonical tissues. Further, we sought to replicate the results of a major study which demonstrated the presence of FSHRs in the endothelial cells of perineoplastic blood vessels. METHODS: Immunostaining was performed on 16 surgically excised benign and malignant tumor tissue samples using both monoclonal and polyclonal anti-FSHR antibodies. RESULTS: Positive staining of FSHRs was heterogeneous among the tissue samples used for analysis, and was confirmed not only in tumour and endothelial cells of perineoplastic blood vessels, but also in benign and normal cells. Based on our findings, FSHR staining using a polyclonal antibody appeared to be highly sensitive, but with a relatively low specificity. Comparatively, immunoreactivity using a monoclonal antibody appeared to show high specificity, but relatively low sensitivity. Although the selected monoclonal antibody for FSHRs seemed to be more specific than the polyclonal variant, neither exhibited a high overall specificity. Neither of the antibodies assessed in the present research could replicate the results of the aforementioned major study. CONCLUSIONS: In conclusion, neither of the two commercially available antibodies seem to be appropriate for investigating FSHRs in non-canonical tissues and, by extension, their role in carcinogenesis.
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Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/metabolismo , Inmunohistoquímica/métodos , Neoplasias/diagnóstico , Receptores de HFE/metabolismo , Humanos , Neoplasias/inmunología , Neoplasias/metabolismo , Pronóstico , Receptores de HFE/inmunologíaRESUMEN
Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class.
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Biblioteca de Péptidos , Receptores de HFE/antagonistas & inhibidores , Receptores de HFE/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Animales , Especificidad de Anticuerpos , AMP Cíclico/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Células HEK293 , Humanos , Inmunización , Células L , Masculino , Ratones , Dominios Proteicos , Receptores de HFE/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal , SolubilidadRESUMEN
PURPOSE: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. EXPERIMENTAL DESIGN: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. RESULTS: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1-dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor-transduced T cells away from targeted tumor cells. CONCLUSIONS: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441-53. ©2016 AACR.
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Inmunoterapia , Neoplasias Ováricas/terapia , Receptores de HFE/inmunología , Linfocitos T/inmunología , Animales , Ascitis/inmunología , Ascitis/patología , Exosomas/inmunología , Exosomas/patología , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Ratones , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de HFE/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CONTEXT: GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus. OBJECTIVE: The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri. MATERIALS AND METHODS: One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n = 15) were subcutaneously injected with 10, 20, and 40 µg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40 µg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins. RESULTS: Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p < 0.05), respectively. Uterine weights of TRI-2, TRI-3 reduced by 28.57% and 11.43% (p < 0.05), respectively. The uterine cavity in CET-1, CET-2, and CET-3 increased; the uterine wall became thick. The cytoplasm of endometrial epithelial cells (EEC) increased slightly. In TRI group, the uterine wall thinned. Uterine cavity became narrow slightly in TRI-1. Numbers of uterine glands reduced. The endometrium epithelial thickness (EET) in CET-1 and CET-2 increased by 68.21% and 79.46% (p < 0.05), respectively. EET in TRI-1 was decreased by 13.69%. Uterine wall thicknesses (UWT) in CET-1 and CET-2 were higher than CG, with the increment of 28.59% and 30.72%. UWT of TRI-1, TRI-2, and TRI-3 reduced by 29.35, 15.36, and 14.41%, respectively. Expressions of ESR1, FSHR, and LHR mRNAs in CET and TRI mice increased. ESR1 and FSHR protein levels increased in all experimental mice (p < 0.05), with a maximum of TRI-3. LHR protein levels of the CET decreased. LHR protein levels of TRI group increased, with a maximum of TRI-3 (p < 0.05). ESR1 protein level had significant negative correlations to mRNA expressions of ESR1, LHR, and FSHR. CONCLUSIONS: CET immunity promoted the uterine development, improved EET and UWT, and also promoted the expressions of ESR1 and FSHR protein levels. It lessened the LHR protein levels. TRI immunity blocked EET and UWT, inhibited uterine growth and development. The efficacy of CET immunity was more obvious than TRI.
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Receptor alfa de Estrógeno/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Receptores de HFE/biosíntesis , Receptores de HL/biosíntesis , Pamoato de Triptorelina/farmacología , Útero/crecimiento & desarrollo , Animales , Receptor alfa de Estrógeno/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Hormona Liberadora de Gonadotropina/farmacología , Ratones , Receptores de HFE/inmunología , Receptores de HL/inmunología , Útero/inmunologíaRESUMEN
Nanovehicles are promising delivery systems for various vaccines. Nevertheless, different biophysicochemical properties of nanoparticles (NPs), dominating their in vitro and in vivo performances for vaccination, remain unclear. We attempted to elucidate the effects of NPs and their pH-sensitivity on in vitro and in vivo efficacy of resulting prophylactic nanovaccines containing a contraceptive peptide (FSHR). To this end, pH-responsive and non-responsive nanovaccines were produced using acetalated ß-cyclodextrin (Ac-bCD) and poly(lactic-co-glycolic acid) (PLGA), respectively. Meanwhile, FSHR derived from an epitope of the follicle-stimulating hormone receptor was used as the model antigen. FSHR-containing Ac-bCD and PLGA NPs were successfully prepared by a nanoemulsion technique, leading to well-shaped nanovaccines with high loading efficiency. The pH-sensitivity of Ac-bCD and PLGA nanovaccines was examined by in vitro hydrolysis and antigen release studies. Nanovaccines could be effectively engulfed by dendritic cells (DCs) via endocytosis in both dose and time dependent manners, and their intracellular trafficking was closely related to the pH-sensitivity of the carrier materials. Furthermore, nanovaccines could induce the secretion of inflammatory cytokines by DCs and T cells co-cultured with the stimulated DCs. In vivo evaluations demonstrated that nanovaccines were more potent than that based on the complete Freund's adjuvant, with respect to inducing anti-FSHR antibody, reducing the sperm count, inhibiting the sperm motility, and increasing the teratosperm rate. Immunization of male mice with nanovaccines notably decreased the parturition incidence of the mated females. Consequently, both in vitro and in vivo activities of FSHR could be considerably augmented by NPs. More importantly, our studies indicated that the pH-responsive nanovaccine was not superior over the non-responsive counterpart for the examined peptide antigen.
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Anticonceptivos/administración & dosificación , Células Dendríticas/efectos de los fármacos , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Receptores de HFE/inmunología , Vacunas/administración & dosificación , Animales , Anticonceptivos/química , Anticonceptivos/farmacología , Citocinas/inmunología , Células Dendríticas/inmunología , Liberación de Fármacos , Femenino , Fertilidad/efectos de los fármacos , Concentración de Iones de Hidrógeno , Hidrólisis , Inmunoglobulina G/sangre , Ácido Láctico/administración & dosificación , Ácido Láctico/química , Masculino , Ratones Endogámicos C57BL , Nanopartículas/química , Péptidos/química , Péptidos/farmacología , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Receptores de HFE/química , Recuento de Espermatozoides , Motilidad Espermática , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Vacunas/química , Vacunas/farmacología , beta-Ciclodextrinas/administración & dosificación , beta-Ciclodextrinas/químicaRESUMEN
Adoptive transfer of T cells engineered to express chimeric immunoreceptors is an effective strategy to treat hematologic cancers; however, the use of this type of therapy for solid cancers, such as ovarian cancer, remains challenging because a safe and effective immunotherapeutic target has not yet been identified. Here, we constructed and evaluated a novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR), a highly conserved molecule in vertebrate animals with expression limited to gonadal tissues, ovarian cancer, and cancer-associated vasculature. Receptor ligand-based anti-FSHR immunoreceptors were constructed that contained small binding fragments from the ligand for FSHR, FSH, fused to T-cell transmembrane and T-cell signaling domains. Human T cells transduced to express anti-FSHR immunoreceptors were specifically immunoreactive against FSHR-expressing human and mouse ovarian cancer cell lines in an MHC-nonrestricted manner and mediated effective lysis of FHSR-expressing tumor cells, but not FSHR-deficient targets, in vitro. Similarly, the outgrowth of human ovarian cancer xenografts in immunodeficient mice was significantly inhibited by the adoptive transfer of FSHR-redirected T cells. Our experimental observations show that FSHR is a promising immunotherapeutic target for ovarian cancer and support further exploration of FSHR-targeted immune therapy approaches for patients with cancer.
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Inmunoterapia Adoptiva , Neoplasias/genética , Neoplasias/inmunología , Receptores de HFE/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Ratones , Neoplasias/terapia , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Receptores de HFE/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PROBLEM: In our previous study on adult male mice, we had identified one immunodominant epitope in hEppin and three epitopes in hFSHR that caused fertility inhibition. But it only demonstrated a moderate inhibitory effect on fertility, and the antifertility effect was unsatisfactory. METHOD OF STUDY: Based on the protein prime-peptide boost inoculation modalities, we further investigated whether the antifertility capacity could be enhanced by a combined immunization with the two antigens. RESULTS: The results displayed a enhanced suppressed fertility (F2EP2C 6.67%) in male mice similar to that seen after four separate administrations of the two proteins (F12E-4 5%). The most likely mechanism by which this antifertility efficacy was achieved was probably through the production of antibodies that led not only to impairment of spermatogenesis but also to inhibition of sperm motility. Moreover, this treatment also induced high concentrations of neutralizing antibodies which were secreted into the lumen of the epididymis. CONCLUSION: Thus, a combination immunization with hFSHR and hEppin enhanced the contraceptive effects and may provide a better means of immunocontraception.
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Anticoncepción Inmunológica/métodos , Fertilidad/inmunología , Proteínas Inhibidoras de Proteinasas Secretoras/inmunología , Receptores de HFE/inmunología , Animales , Linfocitos B/inmunología , Epítopos/inmunología , Femenino , Humanos , Inmunización , Inmunoglobulina A/sangre , Inmunoglobulina G/inmunología , Hormona Luteinizante/sangre , Masculino , Ratones Endogámicos BALB C , Péptidos/inmunología , Motilidad Espermática , Testículo/patología , Testosterona/sangreRESUMEN
Characterizing anti-drug antibodies for neutralizing activity is commonly part of the immunogenicity testing package for most therapeutic proteins. Cell-based neutralization assays can generally be categorized as direct- or indirect assays depending on whether they are associated with therapeutics with agonistic- or antagonistic properties. This paper's aim is a comparison of the two direct neutralization assay formats; the variable- and fixed concentration assay format, using recombinant follicle-stimulating hormone as drug agonist. Essential validation- and performance parameters, such as sample through-put, cut-point, precision, sensitivity and drug tolerance, were compared. The fixed concentration assay format offers superior sample through-put (40 versus 6 samples), precision (coefficient of variation of ≤14% versus 34%) and almost 6 times better sensitivity and is generally recommended as the better option particularly for quasi-quantitative assessments of neutralizing antibodies.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Pruebas de Neutralización/métodos , Receptores de HFE/agonistas , Animales , Anticuerpos Bloqueadores/metabolismo , Unión Competitiva , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Variaciones Dependientes del Observador , Receptores de HFE/genética , Receptores de HFE/inmunología , Sensibilidad y Especificidad , Transgenes/genéticaRESUMEN
OBJECTIVE: To prepare camel derived nanobodies which specifically bind to follicle-stimulating hormone receptor (FSHR). METHODS: The FSHR gene fragment (fshr234) was expressed in E.coli as antigen for affinity screening against VHH phage display library constructed from Xinjiang Bactrian camel. After confirmed by DNA sequencing, the vhh gene fragments of interest were subcloned into pET30a expression vector, and then were used to transform E.coli BL21(DE3). After IPTG induction, 6×His and c-Myc tagged fusion nanobodies were expressed. The nanobodies were purified by Ni-ion affinity chromatography. The binding specificity of nanobodies with His-FSHR234 was determined by ELISA. RESULTS: By enrichment screening with the antigen His-FSHR234, the 28 clones showed VHH sequence identities in DNA sequencing from 40 randomly selected binding clones. The 4 clones were subcloned into pET30a vector and confirmed as expected size of inserts by PCR and endonuclease digestion. The 4 expressed and affinity purified recombinant nanobodies namely VHHFSHR;-06, VHHFSHR;-25, VHHFSHR;-30 and VHHFSHR;-50 showed single band at Mr; 31 000, 26 000, 25 000 and 26 000 on SDS-PAGE, respectively. ELISA results showed that 4 nanobodies could bind to FSHR234 specifically, in which VHHFSHR;-06 showed the highest antigen binding activity. CONCLUSION: By screening camel VHH phage display library with His-FSHR234 antigen, one nanobody, VHHFSHR;-06 with relatively high antigen binding activity has been produced and identified.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de HFE/inmunología , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Camelus/genética , Camelus/inmunología , Camelus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Datos de Secuencia Molecular , Receptores de HFE/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismoRESUMEN
In a previous study it was found that priming with recombinant human follicle-stimulating hormone receptor (rhFSHR) protein (F140) and boosting with a peptide containing amino acids 32-44 from FSHR showed a specific immune response and fertility inhibition in adult male mice. However, this priming and boosting led to damage of the reproductive organs. Therefore, to eliminate this damage, the peptide prime-boost strategy was explored as a possible means of avoiding the pathological change while maintaining infertility. Immunisation with the peptide prime-boost strategy led to decreased fertility 10 weeks after vaccination, which is consistent with Balb/C mice treated with the protein prime-peptide boost regime. In contrast to the cellular swelling and spotty necrosis in spermatogonia observed in the protein-primed mice, the mice receiving peptide priming did not display pathological damage in seminiferous tubules and interstitial cells. Thus, the prime-boost immune regime with the FSHR-derived peptide potentially provides a much safer candidate for a contraceptive vaccine.
Asunto(s)
Genitales Masculinos/efectos de los fármacos , Infertilidad Masculina/inducido químicamente , Receptores de HFE/inmunología , Vacunas Anticonceptivas/efectos adversos , Vacunas Anticonceptivas/farmacología , Animales , Femenino , Enfermedades de los Genitales Masculinos/epidemiología , Enfermedades de los Genitales Masculinos/etiología , Genitales Masculinos/patología , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Receptores de HFE/química , Análisis de Semen , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/inmunologíaRESUMEN
BACKGROUND: In adult humans, the follicle-stimulating hormone (FSH) receptor is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels. METHODS: We used immunohistochemical and immunoblotting techniques involving four separate FSH-receptor-specific monoclonal antibodies that recognize different FSH receptor epitopes and in situ hybridization to detect FSH receptor in tissue samples from patients with a wide range of tumors. Immunoelectron microscopy was used to detect FSH receptor in mouse tumors. RESULTS: In all 1336 patients examined, FSH receptor was expressed by endothelial cells in tumors of all grades, including early T1 tumors. The tumors were located in the prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary. In specimens obtained during surgery performed to remove tumors, the FSH receptor was not expressed in the normal tissues located more than 10 mm from the tumors. The tumor lymphatic vessels did not express FSH receptor. The endothelial cells that expressed FSH receptor were located at the periphery of the tumors in a layer that was approximately 10 mm thick; this layer extended both into and outside of the tumor. Immunoelectron microscopy in mice with xenograft tumors, after perfusion with antiFSH-receptor antibodies coupled to colloidal gold, showed that the FSH receptor is exposed on the luminal endothelial surface and can bind and internalize circulating ligands. CONCLUSIONS: FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).
Asunto(s)
Células Endoteliales/química , Neoplasias/irrigación sanguínea , Neoplasias/química , Neoplasias de la Próstata/irrigación sanguínea , Receptores de HFE/análisis , Animales , Anticuerpos Monoclonales/análisis , Vasos Sanguíneos/química , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas Inmunológicas , Hibridación in Situ , Masculino , Ratones , Microscopía Inmunoelectrónica , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/química , Neoplasias de la Próstata/química , Receptores de HFE/inmunología , Trasplante HeterólogoAsunto(s)
Autoanticuerpos/sangre , Gónadas/inmunología , Infertilidad/diagnóstico , Insuficiencia Ovárica Primaria/diagnóstico , Espermatozoides/inmunología , Zona Pelúcida/inmunología , Biomarcadores/sangre , Femenino , Humanos , Inmunoensayo/métodos , Pruebas de Fijación de Látex/métodos , Masculino , Receptores de HFE/inmunología , Receptores de HL/inmunología , Manejo de Especímenes/métodosRESUMEN
The follicle stimulating hormone (FSH) is of great importance in reproduction modulation of both sexes. The extracellular domain (ECD) of its receptor (FSHR) is crucial for FSH binding and subsequent signal transduction; therefore, it is the potential target for fertility control. To avoid unwanted side-effect when used as immunocontraceptive agent, the ECD was analysed by online prediction combined with molecular docking to identify the candidate B-cell epitopes. Four potential B-cell epitopes were identified and synthesised in tandem with Pan DR epitope. Then the epitope-based peptides were used to boost adult male mice following rhFSHR protein priming, thus to determine their immune responses and fertility inhibition capacity. Three of the four peptides showed suppressed fertility accompanied with small testis and lower serum testosterone level, which was consistent with absolutely lower sperm quantity and poor quality. Among the four epitope peptides, Pep2 displayed the lowest fertility rate of 26.67%, which was similar to that of rhFSHR homologously prime/boost mice (23.30 and 25.00%). Thus, we identified a novel immunodominant B-cell epitope by molecular docking and protein prime/peptide boost strategy.
Asunto(s)
Epítopos de Linfocito B/inmunología , Fertilidad/inmunología , Receptores de HFE/inmunología , Análisis de Varianza , Animales , Western Blotting , Copulación , Ensayo de Inmunoadsorción Enzimática , Epidídimo/inmunología , Epidídimo/metabolismo , Epítopos de Linfocito B/metabolismo , Hormona Folículo Estimulante/inmunología , Hormona Folículo Estimulante/metabolismo , Epítopos Inmunodominantes/inmunología , Hormona Luteinizante/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Radioinmunoensayo , Receptores de HFE/metabolismo , Recuento de Espermatozoides , Coloración y Etiquetado , Testículo/inmunología , Testículo/metabolismo , Testosterona/sangre , VacunaciónRESUMEN
Structure-function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction between the hormone and the receptor is complex and not well understood. In vitro studies using synthetic peptides from the extracellular domain of the receptor and corresponding antipeptide antibodies have suggested that the 285-309 region is surface-oriented. Antipeptide antibodies to this region also inhibit hormone-receptor interaction in a dose-dependent manner and the mechanism of inhibition appears to be competitive in nature. To test this hypothesis in an animal model, antibodies to peptide 285-309 from rat follicle stimulating hormone receptor (FSHR) were developed and characterized. These antibodies were able to detect FSHR in rat ovaries by immunohistochemistry. Further, these antibodies were administered into adult female rats and their effect on fertility status was monitored. These antibodies were found to neutralize the biological activity of endogenous receptor, which resulted in the induction of infertility in the treated animals. Thus, bioneutralization of FSHR has been achieved by targeting its region 285-309 in an in vivo system.