RESUMO
BACKGROUND: Use of oral live-attenuated polio vaccines (OPV), and injected inactivated polio vaccines (IPV) has almost achieved global eradication of wild polio viruses. To address the goals of achieving and maintaining global eradication and minimising the risk of outbreaks of vaccine-derived polioviruses, we tested novel monovalent oral type-2 poliovirus (OPV2) vaccine candidates that are genetically more stable than existing OPVs, with a lower risk of reversion to neurovirulence. Our study represents the first in-human testing of these two novel OPV2 candidates. We aimed to evaluate the safety and immunogenicity of these vaccines, the presence and extent of faecal shedding, and the neurovirulence of shed virus. METHODS: In this double-blind, single-centre phase 1 trial, we isolated participants in a purpose-built containment facility at the University of Antwerp Hospital (Antwerp, Belgium), to minimise the risk of environmental release of the novel OPV2 candidates. Participants, who were recruited by local advertising, were adults (aged 18-50 years) in good health who had previously been vaccinated with IPV, and who would not have any contact with immunosuppressed or unvaccinated people for the duration of faecal shedding at the end of the study. The first participant randomly chose an envelope containing the name of a vaccine candidate, and this determined their allocation; the next 14 participants to be enrolled in the study were sequentially allocated to this group and received the same vaccine. The subsequent 15 participants enrolled after this group were allocated to receive the other vaccine. Participants and the study staff were masked to vaccine groups until the end of the study period. Participants each received a single dose of one vaccine candidate (candidate 1, S2/cre5/S15domV/rec1/hifi3; or candidate 2, S2/S15domV/CpG40), and they were monitored for adverse events, immune responses, and faecal shedding of the vaccine virus for 28 days. Shed virus isolates were tested for the genetic stability of attenuation. The primary outcomes were the incidence and type of serious and severe adverse events, the proportion of participants showing viral shedding in their stools, the time to cessation of viral shedding, the cell culture infective dose of shed virus in virus-positive stools, and a combined index of the prevalence, duration, and quantity of viral shedding in all participants. This study is registered with EudraCT, number 2017-000908-21 and ClinicalTrials.gov, number NCT03430349. FINDINGS: Between May 22 and Aug 22, 2017, 48 volunteers were screened, of whom 15 (31%) volunteers were excluded for reasons relating to the inclusion or exclusion criteria, three (6%) volunteers were not treated because of restrictions to the number of participants in each group, and 30 (63%) volunteers were sequentially allocated to groups (15 participants per group). Both novel OPV2 candidates were immunogenic and increased the median blood titre of serum neutralising antibodies; all participants were seroprotected after vaccination. Both candidates had acceptable tolerability, and no serious adverse events occurred during the study. However, severe events were reported in six (40%) participants receiving candidate 1 (eight events) and nine (60%) participants receiving candidate 2 (12 events); most of these events were increased blood creatinine phosphokinase but were not accompanied by clinical signs or symptoms. Vaccine virus was detected in the stools of 15 (100%) participants receiving vaccine candidate 1 and 13 (87%) participants receiving vaccine candidate 2. Vaccine poliovirus shedding stopped at a median of 23 days (IQR 15-36) after candidate 1 administration and 12 days (1-23) after candidate 2 administration. Total shedding, described by the estimated median shedding index (50% cell culture infective dose/g), was observed to be greater with candidate 1 than candidate 2 across all participants (2·8 [95% CI 1·8-3·5] vs 1·0 [0·7-1·6]). Reversion to neurovirulence, assessed as paralysis of transgenic mice, was low in isolates from those vaccinated with both candidates, and sequencing of shed virus indicated that there was no loss of attenuation in domain V of the 5'-untranslated region, the primary site of reversion in Sabin OPV. INTERPRETATION: We found that the novel OPV2 candidates were safe and immunogenic in IPV-immunised adults, and our data support the further development of these vaccines to potentially be used for maintaining global eradication of neurovirulent type-2 polioviruses. FUNDING: Bill & Melinda Gates Foundation.
Assuntos
Imunogenicidade da Vacina , Vacina Antipólio Oral/efeitos adversos , Vacina Antipólio Oral/imunologia , Poliovirus/imunologia , Adulto , Anticorpos Antivirais/sangue , Método Duplo-Cego , Fezes/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Poliomielite/prevenção & controle , Vacina Antipólio Oral/administração & dosagem , RNA Viral/análise , Método Simples-Cego , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Virulência/imunologia , Eliminação de Partículas Virais/imunologia , Adulto JovemRESUMO
The live-attenuated oral poliovirus vaccine (OPV or Sabin vaccine) replicates in gut-associated tissues, eliciting mucosa and systemic immunity. OPV protects from disease and limits poliovirus spread. Accordingly, vaccination with OPV is the primary strategy used to end the circulation of all polioviruses. However, the ability of OPV to regain replication fitness and establish new epidemics represents a significant risk of polio re-emergence should immunization cease. Here, we report the development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. We introduced modifications within at the 5' untranslated region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region to prevent recombination, and 3D polymerase to limit viral adaptability. Prior work established that nOPV2 is immunogenic in preclinical and clinical studies, and thus may enable complete poliovirus eradication.
Assuntos
Engenharia Genética/métodos , Vacina Antipólio Oral/genética , Vacina Antipólio Oral/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Adulto , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Imunogenicidade da Vacina , Masculino , Camundongos , Poliomielite/prevenção & controle , Poliovirus/genética , Vacina Antipólio Oral/administração & dosagem , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Recombinação Genética , Vacinação , Células Vero , VirulênciaRESUMO
Although global polio eradication is within reach, sustained eradication of all polioviruses requires cessation of oral poliovirus vaccine use to mitigate against vaccine-derived poliovirus circulation and vaccine-associated paralytic poliomyelitis. The first step in this direction was the WHO-recommended global withdrawal of live attenuated type 2 Sabin poliovirus from routine immunisation in May 2016, with future use restricted to outbreak response, and handling controlled by strict containment provisions (GAPIII). This creates unique challenges for development and testing of novel type 2 poliovirus vaccines. We describe the creation of a novel purpose-built containment facility, Poliopolis, to study new monovalent OPV2 vaccine candidates in healthy adult volunteers, which may be a model for future endeavors in vaccine development for emergency use.
Assuntos
Contenção de Riscos Biológicos/métodos , Erradicação de Doenças/métodos , Saúde Global , Habitação , Poliomielite/prevenção & controle , Vacina Antipólio Oral/administração & dosagem , Adulto , Surtos de Doenças/prevenção & controle , Fezes/virologia , Voluntários Saudáveis , Humanos , Poliovirus , Vacina Antipólio Oral/imunologia , Quarentena , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Eliminação de Partículas Virais , Organização Mundial da SaúdeRESUMO
UNLABELLED: Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, (99m)Tc-7C12 and (99m)Tc-7D12. METHODS: Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). RESULTS: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-7C12, but both showed short plasma half-lives (<10 min). CONCLUSION: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, (99m)Tc-7C12 was selected for further studies.
Assuntos
Receptores ErbB/imunologia , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias/imunologia , Compostos de Organotecnécio/química , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tomografia Computadorizada por Raios X/métodos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Camelídeos Americanos , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Neoplasias/metabolismo , Distribuição TecidualRESUMO
PURPOSE: Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. METHODS: Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with (99m)Tc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of (99m)Tc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR. RESULTS: FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of (99m)Tc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6+/-0.9%IA) compared to total liver uptake (18.9+/-0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+/-0.5%IA/cm(3)) of (99m)Tc-8B6 Nanobody compared to DU145 xenografts (1.8+/-0.3%IA/cm(3), p<0.001). CONCLUSION: The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with (99m)Tc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.
Assuntos
Receptores ErbB/análise , Receptores ErbB/metabolismo , Nanoestruturas , Tecnécio , Tomografia Computadorizada por Raios X/métodos , Animais , Linhagem Celular , Receptores ErbB/química , Humanos , Camundongos , Camundongos Nus , Compostos Radiofarmacêuticos , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Toll-like receptor (TLR) triggering is an important step in the induction of T helper (Th) type 1 T cells which are key players in protection against the intracellular pathogen Mycobacterium (M.) tuberculosis. Here we report on the construction of a fusion protein consisting of a tuberculosis vaccine candidate mycolyl-transferase antigen 85A (Ag85A, Rv3804c) coupled to the outer membrane lipoprotein I (OprI) from Pseudomonas (P.) aeruginosa, a documented TLR2/TLR4 trigger. Subcutaneous boosting with this fusion protein in the absence of adjuvant increased significantly the Ag85A-specific humoral but not cellular immune responses of Ag85A-DNA vaccinated mice. Intranasal priming of C57BL/6 mice with live, attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine, followed by intranasal boosting with OprI-Ag85A increased systemic and local antigen-specific interferon (IFN)-gamma and interleukin (IL)-2 responses in spleen, draining cervical and mediastinal lymph nodes and particularly in lung tissue, as compared to responses in mice only vaccinated with BCG vaccine. Despite enhanced immune responses, boosting with OprI-Ag85A did not increase protective efficacy against M. tuberculosis of either plasmid DNA or BCG vaccine in this experimental setting.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Imunização Secundária , Lipoproteínas/imunologia , Pseudomonas aeruginosa/imunologia , Receptores Toll-Like/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/imunologia , Aciltransferases/genética , Administração Intranasal , Animais , Antígenos de Bactérias/genética , Vacina BCG/imunologia , Proteínas de Bactérias/genética , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Lipoproteínas/genética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Tuberculose/imunologia , Vacinas de DNA/administração & dosagemRESUMO
Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae beta-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody-enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.
Assuntos
Adenocarcinoma/terapia , Anticorpos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/terapia , Imunização Passiva/métodos , Imunoconjugados/uso terapêutico , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Antígeno Carcinoembrionário/biossíntese , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Feminino , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Nanotecnologia , Biblioteca de Peptídeos , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , beta-Lactamases/metabolismo , beta-Lactamases/farmacocinética , beta-Lactamases/farmacologiaRESUMO
Secretory leukocyte protease inhibitor (SLPI), an epithelial-specific serine protease inhibitor of the whey acidic protein (WAP) family, exerts broad tissue-protective functions: on the one hand, it counteracts inflammatory responses and invading pathogens, and on the other hand, it repairs the damaged tissue. Here, we report that subcutaneous in vivo passage of low-malignant 3LL-S cells increases the malignancy of these cancer cells. Applying the subtraction suppressive hybridization method to this cancer model, we identify SLPI as one of the genes whose level of expression directly correlates with the malignancy of the cancer cells. Using transfection experiments, we demonstrate that overexpression of mouse or human SLPI in 3LL-S cells is sufficient to enhance their malignant behavior, and that this activity of SLPI is related to its ability to inhibit serine proteases. Furthermore, using cDNA dot-blot hybridization, we show that in human gynecological cancer tissue SLPI expression is frequently increased as compared with normal tissue. This study underscores the promalignant role of SLPI in the development of cancer and its potential usefulness as a biomarker for gynecological cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas/fisiologia , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Hibridização de Ácido Nucleico , Proteínas Secretadas Inibidoras de Proteinases , Inibidor Secretado de Peptidases Leucocitárias , Serina Endopeptidases/metabolismo , Distribuição Tecidual , Transfecção , Regulação para CimaRESUMO
UNLABELLED: Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain-only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-(125/131)I-iodobenzoate (*I-SGMIB). METHODS: The 5F7GGC Nanobody was radiolabeled using *I-SGMIB and, for comparison, with N(ε)-(3-*I-iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-GEEEK (*I-IB-Mal-d-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN ((125)I-Nanobody). The 3 labeled Nanobodies were evaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts. RESULTS: *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated (125)I-Nanobody or *I-IB-Mal-d-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor-to-normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs and kidneys, for which the values were 10.4 ± 4.5 and 5.2 ± 1.5, respectively. CONCLUSION: Labeling anti-HER2 Nanobody 5F7GGC with *I-SGMIB yields a promising new conjugate for targeting HER2-expressing malignancies. Further research is needed to determine the potential utility of *I-SGMIB-5F7GGC labeled with (124)I, (123)I, and (131)I for PET and SPECT imaging and for targeted radiotherapy, respectively.
Assuntos
Benzoatos , Guanidina/análogos & derivados , Compostos Radiofarmacêuticos , Receptor ErbB-2/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzoatos/síntese química , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Feminino , Guanidina/síntese química , Humanos , Marcação por Isótopo , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. METHODS: 5F7GGC Nb was radioiodinated with ¹²5I using Iodogen and with ¹³¹I using the residualizing agent N(É)-(3-[¹³¹I]iodobenzoyl)-Lys5-N(α)-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-D-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. RESULTS: The radiochemical yields for Iodogen and [¹³¹I]IB-Mal-D-GEEEK labeling were 83.6±5.0% (n=10) and 59.6±9.4% (n=15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-D-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65±0.61% ID/g vs. 2.92±0.24% ID/g at 8h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. CONCLUSIONS: Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-D-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²9I) and PET imaging (¹²4I) of patients with HER2-expressing tumors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Terapia de Alvo Molecular , Receptor ErbB-2/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Especificidade de Anticorpos , Transporte Biológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Radioisótopos do Iodo , Maleimidas/química , Camundongos , Transporte Proteico , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/uso terapêuticoRESUMO
Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (αHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two αHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 ((89)Zr, t(1/2;) = 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 µg per mouse. The (89)Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g ± 0.6%ID/g, 7.2%ID/g ± 1.0%ID/g, 3.4%ID/g ± 0.3%ID/g, and 0.3%ID/g ± 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g ± 1.1%ID/g, 8.9%ID/g ± 1.0%ID/g, 8.7%ID/g ± 1.5%ID/g, and 7.2%ID/g ±1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-µg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGF-expressing tumors.
Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Fator de Crescimento de Hepatócito/metabolismo , Compostos Radiofarmacêuticos/farmacologia , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/farmacocinética , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio/farmacocinética , Zircônio/farmacologiaRESUMO
Evolution has been continuously honing the design of antibodies to function as specific molecular markers that are able to alert the immune system to the presence of pathogenic antigens, and to recruit complement- and Fc receptor-bearing effector cells. During the past 25 years, the versatility of antibodies has been applied to several therapeutic applications. The development of new technologies, combined with data obtained using a new generation of antibody reagents, have allowed the adaptation of the design of antibodies to better match drug development requirements. Nanobodies are therapeutic proteins derived from the heavy-chain variable (VHH) domains that occur naturally in heavy-chain-only Ig molecules in camelidae. These VHH domains are the smallest known antigen-binding antibody fragments. Nanobodies can be easily produced in prokaryotic or eukaryotic host organisms, and their unique biophysical and pharmacological characteristics render these molecules ideal candidates for drug development. This review describes the structural properties of nanobodies and focuses on their unique features, which distinguishes these molecules from other antibody formats and small-molecule drugs. Possible therapeutic applications of nanobodies are discussed and data from phase I clinical trials of the novel 'first-in-class' anti-thrombotic agent ALX-0081 (Ablynx NV) are presented.
Assuntos
Desenho de Fármacos , Fragmentos de Imunoglobulinas/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/imunologia , Anticorpos de Domínio Único/uso terapêutico , Animais , Anticorpos/imunologia , Camelídeos Americanos , Ensaios Clínicos Fase I como Assunto , Fibrinolíticos/imunologia , Fibrinolíticos/farmacologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Anticorpos de Domínio Único/imunologiaRESUMO
The development of mucosal vaccines requires antigen delivery and adjuvant systems that can efficiently help in presenting vaccine antigens to the mucosal immune system. The outer membrane lipoprotein I (OprI) of Pseudomonas aeruginosa seems to possess both the quality to induce a non-specific immune response (adjuvant effect through its lipid tail) as well as the quality to facilitate uptake of the vaccine antigen by interacting with Toll-like receptor 2/4 (TLR2/4) on antigen-presenting cells (APC) and epithelial cells (adhesion effect). Here, we show for the first time the adhesion of OprI to epithelial cells of the trachea and small intestine of chickens. Adhesion could be seen on cryosections after in vitro as well as after in vivo incubation of the trachea and intestine. This proves the value of OprI as a fusion partner in mucosal protein vaccine development, which is especially important for poultry where mass vaccination is only possible by the respiratory or oral route.
Assuntos
Proteínas de Bactérias/metabolismo , Mucosa Intestinal/metabolismo , Lipoproteínas/metabolismo , Pseudomonas aeruginosa/química , Traqueia/metabolismo , Animais , Proteínas de Bactérias/isolamento & purificação , Galinhas , Imuno-Histoquímica , Lipoproteínas/isolamento & purificação , Microscopia de Fluorescência , Mucosa/metabolismo , Ligação ProteicaRESUMO
The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from "immune" phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.
Assuntos
Anticorpos/farmacologia , Receptores ErbB/antagonistas & inibidores , Cadeias Pesadas de Imunoglobulinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos/isolamento & purificação , Formação de Anticorpos , Especificidade de Anticorpos , Camelídeos Americanos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Ligantes , Camundongos , Camundongos Nus , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-gamma ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from naïve animals. In addition to the IFN-gamma responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible--activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Peste Suína Clássica/imunologia , Citotoxicidade Imunológica , Citometria de Fluxo/veterinária , Imunidade Celular/imunologia , Interferon gama/metabolismo , Ativação Linfocitária , Testes de Neutralização/veterinária , RNA Helicases/imunologia , Distribuição Aleatória , Serina Endopeptidases/imunologia , Organismos Livres de Patógenos Específicos , Suínos , Linfócitos T Citotóxicos/imunologia , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Bacterial lipoproteins are potent stimulators of innate immune responses and can mediate humoral and cytotoxic T cell responses without additional adjuvants. OprI derived from Pseudomonas aeruginosa was tested in vitro and in vivo for its adjuvant potential in the context of a classical swine fever (CSF) subunit vaccine. OprI activated porcine monocyte-derived dendritic cells (MoDC), upregulating CD80/86 and MHC class II expression, as well as pro-inflammatory cytokines. OprI enhanced CSFV-antigen-specific lymphocyte proliferation and IFN-gamma release. An E2/NS3-based subunit vaccine adjuvanted with OprI stimulated specific immune responses and partial protection against CSFV infection. Although, a water-oil-water adjuvanted vaccine was more potent at protecting animals, this study demonstrates that OprI has immunostimulatory properties for porcine DC, and has potential as vaccine immunostimulant. Further studies are necessary to optimize antigen formulation enabling to translate the in vitro efficacy into a potent vaccine in vivo.
Assuntos
Adjuvantes Imunológicos/farmacologia , Proteínas de Bactérias/farmacologia , Vírus da Febre Suína Clássica/imunologia , Lipoproteínas/farmacologia , Pseudomonas aeruginosa/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Células Dendríticas/fisiologia , Interferon-alfa/sangue , Interferon gama/biossíntese , Ativação Linfocitária , Fenótipo , Suínos , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
According to the cancer immunoediting concept, inflammatory mediators play not only a critical role in promoting host protection against cancer but also contribute to cancer cell growth and survival. TNF-alpha is a critical factor in this network. However, the mechanisms underlying the tumor-promoting effect of TNF-alpha have not been fully elucidated yet. We previously reported that in vitro culture of Lewis lung carcinoma 3LL cells with TNF-alpha-producing macrophages resulted in enhanced resistance toward TNF-alpha-mediated lysis and increased malignancy of the 3LL cells. In this study, we analyzed the effects of endogenous TNF-alpha on TNF-alpha resistance and malignant behavior in vivo of low-malignant/TNF-alpha-sensitive 3LL-S cells and cancer cells derived from 3LL-S tumors that developed in wild-type or TNF-alpha(-/-) mice. Interestingly, 3LL-S cells acquired a malignant phenotype in vivo depending on the presence of host TNF-alpha, whereas acquisition of TNF-alpha resistance was TNF-alpha-independent. This result suggested that malignancy-promoting characteristics of 3LL-S cells other than TNF-alpha resistance are influenced in vivo by TNF-alpha. We previously identified the malignancy-promoting genes, secretory leukocyte protease inhibitor (SLPI) and S100A4, as being up-regulated in 3LL-S cells upon their s.c. growth in wild-type mice. In this study, we show that SLPI, but not S100A4, was induced in 3LL-S cells both in vitro and in vivo by TNF-alpha, and that silencing of in vivo induced 3LL-S SLPI expression using RNA interference abrogated in vivo progression but did not influence TNF-alpha resistance. These data indicate that SLPI induction may be one mechanism whereby TNF-alpha acts as an endogenous tumor promoter.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Citotoxicidade Imunológica , Invasividade Neoplásica/imunologia , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Animais , Carcinoma Pulmonar de Lewis/metabolismo , Feminino , Expressão Gênica , Camundongos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although activation of Toll-like receptor 4 (TLR4)-positive cells is essential for eliminating Gram-negative bacteria, overactivation of these cells by the TLR4 ligand LPS initiates a systemic inflammatory reaction and shock. Here we demonstrate that SPRET/Ei mice, derived from Mus spretus, exhibit a dominant resistance against LPS-induced lethality. This resistance is mediated by bone marrow-derived cells. Macrophages from these mice exhibit normal signaling and gene expression responses that depend on the myeloid differentiation factor 88 adaptor protein, but they are impaired in IFN-beta production. The defect appears to be specific for IFN-beta, although the SPRET/Ei IFN-beta promoter is normal. In vivo IFN-beta induction by LPS or influenza virus is very low in SPRET/Ei mice, but IFN-beta-treatment restores the sensitivity to LPS, and IFN type 1 receptor-deficient mice are also resistant to LPS. Because of the defective induction of IFN-beta, these mice are completely resistant to Listeria monocytogenes and highly sensitive to Leishmania major infection. Stimulation of SPRET/Ei macrophages leads to rapid down-regulation of IFN type 1 receptor mRNA expression, which is reflected in poor induction of IFN-beta-dependent genes. This finding indicates that the resistance of SPRET/Ei mice to LPS is due to disruption of a positive-feedback loop that amplifies IFN-beta production. In contrast to TLR4-deficient mice, SPRET/Ei mice resist both LPS and sepsis induced with Klebsiella pneumoniae.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células da Medula Óssea/imunologia , Interferon beta/biossíntese , Lipopolissacarídeos/imunologia , Camundongos Endogâmicos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células da Medula Óssea/metabolismo , Suscetibilidade a Doenças , Regulação para Baixo , Retroalimentação Fisiológica , Feminino , Leishmaniose Cutânea/imunologia , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide , Receptor de Interferon alfa e beta , Receptores de Interferon/genética , Receptores de Interferon/metabolismoRESUMO
OBJECTIVE: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA. METHODS: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. RESULTS: The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. CONCLUSION: These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.