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The anti-Müllerian hormone (AMH) belongs to the TGF-ß family and plays a key role during fetal sexual development. Various reports have described the expression of AMH type II receptor (AMHRII) in human gynecological cancers including ovarian tumors. According to qRT-PCR results confirmed by specific In-Situ Hybridization (ISH) experiments, AMHRII mRNA is expressed in an extremely restricted number of normal tissues. By performing ISH on tissue microarray of solid tumor samples AMHRII mRNA was unexpectedly detected in several non-gynecological primary cancers including lung, breast, head and neck, and colorectal cancers. AMHRII protein expression, evaluated by immunohistochemistry (IHC) was detected in approximately 70% of epithelial ovarian cancers. Using the same IHC protocol on more than 900 frozen samples covering 18 different cancer types we detected AMHRII expression in more than 50% of hepato-carcinomas, colorectal, lung, and renal cancer samples. AMHRII expression was not observed in neuroendocrine lung tumor samples nor in non-Hodgkin lymphoma samples. Complementary analyses by immunofluorescence and flow cytometry confirmed the detection of AMHRII on a panel of ovarian and colorectal cancers displaying comparable expression levels with mean values of 39,000 and 50,000 AMHRII receptors per cell, respectively. Overall, our results suggest that this embryonic receptor could be a suitable target for treating AMHRII-expressing tumors with an anti-AMHRII selective agent such as murlentamab, also named 3C23K or GM102. This potential therapeutic intervention was confirmed in vivo by showing antitumor activity of murlentamab against AMHRII-expressing colorectal cancer and hepatocarcinoma Patient-Derived tumor Xenografts (PDX) models.
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AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4+ Th1/Th2 balance towards a Th1 response and activates CD8+ T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.
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BACKGROUND: Angiotensin-converting enzyme 2 is the receptor that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for entry into lung cells. Because ACE-2 may be modulated by angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), there is concern that patients treated with ACEIs and ARBs are at higher risk of coronavirus disease 2019 (COVID-19) pneumonia. AIM: This study sought to analyze the association of COVID-19 pneumonia with previous treatment with ACEIs and ARBs. MATERIALS AND METHODS: We retrospectively reviewed 684 consecutive patients hospitalized for suspected COVID-19 pneumonia and tested by polymerase chain reaction assay. Patients were split into two groups, according to whether (group 1, n = 484) or not (group 2, n = 250) COVID-19 was confirmed. Multivariable adjusted comparisons included a propensity score analysis. RESULTS: The mean age was 63.6 ± 18.7 years, and 302 patients (44%) were female. Hypertension was present in 42.6% and 38.4% of patients in groups 1 and 2, respectively (P = 0.28). Treatment with ARBs was more frequent in group 1 than group 2 (20.7% vs. 12.0%, respectively; odds ratio [OR] 1.92, 95% confidence interval [CI] 1.23-2.98; P = 0.004). No difference was found for treatment with ACEIs (12.7% vs. 15.7%, respectively; OR 0.81, 95% CI 0.52-1.26; P = 0.35). Propensity score-matched multivariable logistic regression confirmed a significant association between COVID-19 and previous treatment with ARBs (adjusted OR 2.36, 95% CI 1.38-4.04; P = 0.002). Significant interaction between ARBs and ACEIs for the risk of COVID-19 was observed in patients aged > 60 years, women, and hypertensive patients. CONCLUSIONS: This study suggests that ACEIs and ARBs are not similarly associated with COVID-19. In this retrospective series, patients with COVID-19 pneumonia more frequently had previous treatment with ARBs compared with patients without COVID-19.
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Bloqueadores del Receptor Tipo 2 de Angiotensina II/efectos adversos , Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , COVID-19/complicaciones , Neumonía/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Bloqueadores del Receptor Tipo 2 de Angiotensina II/uso terapéutico , COVID-19/diagnóstico , Femenino , Humanos , Hipertensión/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Neumonía/diagnóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND: Besides the interest of an early detection of ovarian cancer, there is an urgent need for new predictive and prognostic biomarkers of tumor development and cancer treatment. In healthy patients, circulating blood monocytes are typically subdivided into classical (85%), intermediate (5%) and non-classical (10%) populations. Although these circulating monocyte subsets have been suggested as biomarkers in several diseases, few studies have investigate their potential as a predictive signature for tumor immune status,tumor growth and treatment adaptation. METHODS: In this study, we used a homogeneous cohort of 28 chemotherapy-naïve patients with ovarian cancer to evaluate monocyte subsets as biomarkers of the ascites immunological status. We evaluated the correlations between circulating monocyte subsets and immune cells and tumor burden in peritoneal ascites. Moreover, to validate the use of circulating monocyte subsets tofollow tumor progression and treatment response, we characterized blood monocytes from ovarian cancer patients included in a phase 1 clinical trial at baseline and following murlentamab treatment. RESULTS: We demonstrate here a robust expansion of the intermediate blood monocytes (IBMs) in ovarian cancer patients. We establish a significant positive correlation between IBM percentage and tumor-associate macrophages with a CCR2high/CD163high/CD206high/CD86lowprofile. Moreover, IBM expansion is associated with a decreased effector/regulatory T-cell ratio in ascites and with the presence of soluble immunosuppressive mediators. We also establish that IBM proportion positively correlates with the peritoneum tumor burden. Finally, the study of IBMs in patients with ovarian cancer under immunotherapy during the phase clinical trial supports IBMs to follow the evolution of tumor development and the treatment adaptation. CONCLUSIONS: This study, which links IBM level with immunosuppression and tumor burden in peritoneum, identifies IBMs as apotential predictive signature of ascites immune status and as a biomarker ofovarian cancer development and treatment response. TRIAL REGISTRATION NUMBER: EudraCT: 2015-004252-22 NCT02978755.
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Ascitis/genética , Biomarcadores de Tumor/metabolismo , Inmunoterapia/métodos , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Receptores de IgG/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Microambiente TumoralRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
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Carcinoma Ductal de Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Receptores ErbB/metabolismo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Recurrencia Local de Neoplasia , PronósticoRESUMEN
BACKGROUND: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways. Here, we wanted to determine the mechanisms underlying the pro-apoptotic effect of 9F7-F11. METHODS: Anti-HER3 antibody-induced apoptosis was assessed by western blot, and by flow cytometry measurement of Annexin V/7-AAD-labelled tumor cells (BxPC3, MDA-MB-468 and DU145 cell lines). c-FLIP/ITCH interaction and subsequent degradation/ubiquitination were investigated by co-immunoprecipitation of ITCH-silenced vs scramble control cells. The relationship between ITCH-mediated c-FLIP degradation and antibody-induced apoptosis was examined by western blot and flow cytometry of tumor cells, after ITCH RNA interference or by pre-treatment with ITCH chemical inhibitor chlorimipramine (CI). RESULTS: Following incubation with 9F7-F11, cancer cell apoptosis occurs through activation of caspase-8, - 9 and - 3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Moreover we showed that ubiquitination and proteasomal degradation of the anti-apoptotic protein c-FLIP was mediated by USP8-regulated ITCH recruitment. This effect was abrogated by ITCH- and USP8-specific RNA interference (siRNA), or by the ITCH chemical inhibitor CI. Specifically, ITCH silencing or CI blocked 9F7-F11-induced caspase-8-mediated apoptosis of tumor cells, and restored c-FLIP expression. ITCH-silencing or CI concomitantly abrogated HER3-specific antibody-induced apoptosis of Annexin V/7-AAD-labelled BxPC3 cells. 9F7-F11 favored the extrinsic apoptosis pathway by inducing TRAIL-R2/DR5 upregulation and TRAIL expression that promoted the formation of death-inducing signaling complex (DISC), leading to caspase-8-mediated apoptosis. Incubation with 9F7-F11 also induced BID cleavage, BAX upregulation and BIM expression, which initiated the caspase-9/3-mediated mitochondrial death pathway. The anti-HER3 antibody pro-apoptotic effect occurred concomitantly with downregulation of the pro-survival proteins c-IAP2 and XIAP. CONCLUSIONS: The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.
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Anticuerpos Monoclonales de Origen Murino/metabolismo , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Humanos , Transducción de SeñalRESUMEN
Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.
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Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR.
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Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Biomarcadores de Tumor/inmunología , Neoplasias/tratamiento farmacológico , Neurregulina-1/genética , Receptor ErbB-3/inmunología , Células A549 , Animales , Anticuerpos Antiidiotipos/administración & dosificación , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales de Origen Murino/inmunología , Biomarcadores de Tumor/genética , Proliferación Celular/efectos de los fármacos , Femenino , Transferencia Resonante de Energía de Fluorescencia , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Neurregulina-1/inmunología , Fosforilación , Unión Proteica , Receptor ErbB-3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ovarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10-11 M vs 7.9 × 10-10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial.
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Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Receptores de Péptidos/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Femenino , Glicosilación , Humanos , Ratones Desnudos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Ingeniería de ProteínasRESUMEN
The Laboratoire français du fractionnement et des biotechnologies (LFB), the leading manufacturer of plasma-derived medicinal products in France and 6th worldwide, is strongly involved in the development of therapeutic monoclonal antibodies (mAb). For more than 15 years, LFB has been focusing its research effort on the study of structure-function relationship of antibodies. Its studies on the molecular basis of IgG interaction with the receptors for the Fc portion of IgG (FcgRs) has made it possible to develop antibodies with high antibody-dependent cellular cytotoxicity (ADCC) activity and enhanced affinity to FcgRIII (CD16), both correlated to a glycosylation pattern characterized by a low fucose content. Based on these studies, LFB has developed EMABling, a technological platform for the production of antibodies with enhanced cytotoxicity ability. Two EMABling antibodies recently entered clinical development: LFB-R593, a fully human anti-rhesus D (RhD) antibody, for the prevention of feto-maternal allo-immunization in RhD- women, as a substitute for human polyclonal anti-RhD immunoglobulins, and LFB-R603, a monoclonal antibody directed against CD20, for the treatment of B cell malignancies. LFB investment in bioproduction through the recent acquisition of MAbgène company, a fully integrated French contract biopharmaceutical manufacturing company, allows the production of antibodies to a large GMP scale. As a whole, LFB owns a portfolio of several EMABling antibodies with high therapeutic interest, in line with its public health mission.
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Anticuerpos Monoclonales , Industria Farmacéutica/organización & administración , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Femenino , Francia , Glicosilación , Humanos , Inmunoglobulina G/inmunología , Recién Nacido , Isoanticuerpos/aislamiento & purificación , Isoanticuerpos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Macaca fascicularis , Embarazo , Procesamiento Proteico-Postraduccional , Receptores de IgG/inmunología , Isoinmunización Rh/prevención & control , Globulina Inmune rho(D) , Tecnología FarmacéuticaRESUMEN
BACKGROUND: Chikungunya virus (CHIKV) is a recently reemerged arbovirus responsible for a massive outbreak of infection in the Indian Ocean region and India that has a very significant potential to spread globally because of the worldwide distribution of its mosquito vectors. CHIKV induces a usually self-limited disease in humans that is characterized by fever, arthralgia, myalgia, and rash; however, cases of severe CHIKV infection have recently been described, particularly in adults with underlying condition and neonates born to viremic mothers. METHODS: Human polyvalent immunoglobulins were purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection, and the preventive and curative effects of these immunoglobulins were investigated in 2 mouse models of CHIKV infection that we developed. RESULTS: CHIKV immunoglobulins contain anti-CHIKV antibodies and exhibit a high in vitro neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection in vivo, including in the neonate. CONCLUSIONS: Administration of CHIKV immunoglobulins may constitute a safe and efficacious prevention strategy and treatment for individuals exposed to CHIKV who are at risk of severe infection, such as neonates born to viremic mothers and adults with underlying conditions. These results provide a proof-of-concept for purifying human immunoglobulins from plasma samples from patients in the convalescent phase of an emerging infectious disease for which neither prevention nor treatment is available.
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Infecciones por Alphavirus/tratamiento farmacológico , Virus Chikungunya , Inmunoglobulinas/uso terapéutico , Vacunas Virales/inmunología , Adulto , Infecciones por Alphavirus/prevención & control , Animales , Humanos , Inmunoglobulinas/sangre , Ratones , Ratones Noqueados , Receptores de Interferón/genética , Factores de TiempoRESUMEN
A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD+ red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD+ RBCs. Autologous RhD+ RBCs were sensitized with either Rhophylac or R297 at three different coating percentages (25, 12.5 and 6.25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6.25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FcgammaRIII binding and increased ADCC.
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Anticuerpos Monoclonales/inmunología , Eritrocitos/inmunología , Isoanticuerpos/inmunología , Receptores de IgG/inmunología , Adulto , Anticuerpos Monoclonales/efectos adversos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Hemólisis/genética , Hemólisis/inmunología , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Receptores de IgG/genética , Globulina Inmune rho(D)/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcgamma receptor IIIA (FcgammaRIIIA)/CD16 binding and FcgammaRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcgammaRIIIA-mediated interleukin-2 production by FcgammaRIIIA(+) Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.
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Antígenos CD20/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Receptores de IgG/inmunología , Anciano , Anciano de 80 o más Años , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Interleucina-2/biosíntesis , Células Asesinas Naturales/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Protein tyrosine phosphatases from several microorganisms have been shown to play a role as virulence factors by modifying the phosphorylation/dephosphorylation equilibrium in cells of their host. Two tyrosine phosphatases, MptpA and MptpB, secreted by Mycobacterium tuberculosis, have been identified. Expression of MptpA is upregulated upon infection of monocytes, but its role in host cells has not been elucidated. A eukaryotic expression vector containing the mptpA cDNA has been transfected into macrophages. We report that MptpA reduced phagocytosis of mycobacteria, opsonized zymosan or zymosan, but had no effect on phagocytosis of IgG-coated particles. We also noted that the presence of F-actin at the surface of phagosomes containing opsonized zymosan was significantly increased in cells expressing MptpA. In the presence of recombinant MptpA, the process of actin polymerization at the surface of isolated phagosomes was increased; this was not the case in the presence of the phosphatase-dead mutant MptpA(C11S). MptpA had no effect when IgG-coated particles were present inside isolated phagosomes. These results indicate that, like other tyrosine phosphatases of pathogens, MptpA plays a role in phagocytosis and actin polymerization. However, MptpA had no effect on IgG particles, suggesting that its putative substrate(s) is not linked to the signaling pathways of Fcgamma receptors.
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Actinas/metabolismo , Proteínas Bacterianas/farmacología , Macrófagos/metabolismo , Mycobacterium tuberculosis/patogenicidad , Fagocitosis/efectos de los fármacos , Proteínas Tirosina Fosfatasas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Actinas/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/enzimología , Células 3T3 NIH , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Genoma Bacteriano , Genotipo , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
In bacteria, regulatory phosphorylation of proteins at serine and/or threonine residues by Ser/Thr protein kinase (STPK) is an emerging theme in prokaryotic signaling, particularly since the prediction of the occurrence of several STPKs from genome sequencing and sequence surveys. Here we show that protein PknH possesses an autokinase activity and belongs to the large STPK family found in Mycobacterium tuberculosis. Evidence is presented that PknH can also phosphorylate EmbR, a protein suspected to modulate the level of arabinosyltransferase activity involved in arabinan biosynthesis of arabinogalactan, a key molecule of the mycobacterial cell wall. Interestingly, EmbR possesses an FHA (forkhead-associated) domain, a newly described phosphoprotein recognition domain, which plays an essential role in PknH-EmbR interaction and phosphorylation of EmbR by PknH. It is demonstrated that mutation of each of three particular residues of this FHA domain, Arg312, Ser326, and Asn348, totally abolishes the PknH-mediated phosphorylation of EmbR, thus highlighting the critical role of this domain in the direct interaction between EmbR and PknH.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Proteínas Nucleares/fisiología , Pentosiltransferasa/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Catálisis , Citosol/química , Citosol/metabolismo , Activación Enzimática , Factores de Transcripción Forkhead , Mycobacterium tuberculosis/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Estructura Terciaria de Proteína/fisiología , Serina/metabolismo , Especificidad por Sustrato , Treonina/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli sigma(70) -35 elements but located much closer to the -10 element is important for optimal expression of P1, whereas the sequence at the -35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Daño del ADN , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Mycobacterium tuberculosis/genética , Análisis de Secuencia de ADN , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed. This protein is shown to be a eukaryotic-like (Hanks'-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases. It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain. The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues. Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity. Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed.