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1.
Sci Immunol ; 6(62)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389613

RESUMO

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue-resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161- and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was up-regulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen-specific population with enhanced esophagus homing potential.


Assuntos
Esofagite Eosinofílica/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Linfócitos T CD4-Positivos/imunologia , Esofagite Eosinofílica/patologia , Humanos , Receptores Acoplados a Proteínas G/imunologia , Receptores de Peptídeos/imunologia , Células Th2/imunologia
2.
J Clin Invest ; 131(3)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33529166

RESUMO

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.


Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Estresse do Retículo Endoplasmático/imunologia , Ativação Linfocitária , Mutação de Sentido Incorreto , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia , Substituição de Aminoácidos , Animais , Apoptose/genética , Proteína Coatomer/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Estresse do Retículo Endoplasmático/genética , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Humanos , Camundongos , Camundongos Mutantes , Receptores de Peptídeos/genética , Receptores de Peptídeos/imunologia , Imunodeficiência Combinada Severa/genética
3.
Sci Immunol ; 5(48)2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532834

RESUMO

The orphan chemoattractant receptor GPR15 is important for homing T lymphocytes to the large intestine, thereby maintaining intestinal immune homeostasis. However, the molecular mechanisms underlying the regulation of GPR15 expression remain elusive. Here, we show a central role of the aryl hydrocarbon receptor (Ahr) in promoting GPR15 expression in both mice and human, thus gut homing of T lymphocytes. Mechanistically, Ahr directly binds to open chromatin regions of the Gpr15 locus to enhance its expression. Ahr transcriptional activity in directing GPR15 expression was modulated by two transcription factors, Foxp3 and RORγt, both of which are expressed preferentially by gut regulatory T cells (Tregs) in vivo. Specifically, Foxp3 interacted with Ahr and enhanced Ahr DNA binding at the Gpr15 locus, thereby promoting GPR15 expression. In contrast, RORγt plays an inhibitory role, at least in part, by competing with Ahr binding to the Gpr15 locus. Our findings thus demonstrate a key role for Ahr in regulating Treg intestinal homing under the steady state and during inflammation and the importance of Ahr-RORγt-Foxp3 axis in regulating gut homing receptor GPR15 expression by lymphocytes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Peptídeos/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética
4.
mSphere ; 5(1)2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941807

RESUMO

Protective antigen (PA) is a component of anthrax toxin that can elicit toxin-neutralizing antibody responses. PA is also the major antigen in the current vaccine to prevent anthrax, but stability problems with recombinant proteins have complicated the development of new vaccines containing recombinant PA. The relationship between antigen physical stability and immunogenicity is poorly understood, but there are theoretical reasons to think that this parameter can affect immune responses. We investigated the immunogenicity of anthrax PA, in the presence and absence of the soluble von Willebrand factor A domain of the human form of receptor capillary morphogenesis protein 2 (sCMG2), to elicit antibodies to PA in BALB/c mice. Prior studies showed that sCMG2 stabilizes the 83-kDa PA structure to pH, chemical denaturants, temperature, and proteolysis and slows the hydrogen-deuterium exchange rate of histidine residues far from the binding interface. In contrast to a vaccine containing PA without adjuvant, we found that mice immunized with PA in stable complex with sCMG2 showed markedly reduced antibody responses to PA, including toxin-neutralizing antibodies and antibodies to domain 4, which correlated with fewer toxin-neutralizing antibodies. In contrast, mice immunized with PA in concert with a nonbinding mutant of sCMG2 (D50A) showed anti-PA antibody responses similar to those observed with PA alone. Our results suggest that addition of sCMG2 to a PA vaccine formulation is likely to result in a significantly diminished immune response, but we discuss the multitude of factors that could contribute to reduced immunogenicity.IMPORTANCE The anthrax toxin PA is the major immunogen in the current anthrax vaccine (anthrax vaccine adsorbed). Improving the anthrax vaccine for avoidance of a cold chain necessitates improvements in the thermodynamic stability of PA. We address how stabilizing PA using sCMG2 affects PA immunogenicity in BALB/c mice. Although the stability of PA is increased by binding to sCMG2, PA immunogenicity is decreased. This study emphasizes that, while binding of a ligand retains or improves conformational stability without affecting the native sequence, epitope recognition or processing may be affected, abrogating an effective immune response.


Assuntos
Vacinas contra Antraz/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Imunogenicidade da Vacina , Receptores de Peptídeos/imunologia , Fator de von Willebrand/metabolismo , Animais , Antraz/imunologia , Antraz/prevenção & controle , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fator de von Willebrand/imunologia
5.
Mol Ther ; 28(2): 548-560, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31870622

RESUMO

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.


Assuntos
Neoplasias dos Genitais Femininos/imunologia , Imunoterapia Adotiva , Neoplasias Ovarianas/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Feminino , Neoplasias dos Genitais Femininos/terapia , Humanos , Camundongos , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Neuroinflammation ; 16(1): 137, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277647

RESUMO

BACKGROUND: The lack of effective treatment for Alzheimer's disease (AD) stems mainly from the incomplete understanding of AD causes. Neuroinflammation has emerged as an important component of AD pathology, and a vast number of experimental and clinical data indicated a crucial role for the activation of the innate immune system in disease promotion and symptom progression. METHODS: Clinical examinations of AD patients in a different stage of disease severity in correlation with the measurement of two innate immune reactions, i.e., peripheral blood leukocyte (PBLs) resistance to viral infection (vesicular stomatitis virus, VSV) ex vivo, and cytokines: TNF-α, IFN-γ, IL-1ß, and IL-10, production with enzyme-linked immunosorbent assay (ELISA), have been investigated during this preliminary study before and after 4 weeks of oral treatment with dietary supplement proline-rich polypeptide complex (PRP) (120 µg of PRP/day). The potential effect of PRP on the distribution of PBLs' subpopulations has been specified. RESULTS: We have found a deficiency in innate immune response in AD patients. It was demonstrated for the first time that the degree of PBLs resistance to VSV infection was closely related to the stage of clinical severity of AD. Our study showed significant differences in cytokine production which pointed that in AD patients innate immune mechanisms are impaired. Administration of PRP to our patients increased innate immune response of PBLs and declined pro- and anti-inflammatory cytokine production, thus subduing the excessively developed inflammatory response, especially among patients with high severity of AD. PRP did not exhibit a pro-proliferative activity. It was showed, however, significant influence of PRP on the distribution of PBLs' subpopulations. CONCLUSION: The findings mentioned above might be crucial in the context of potential application of immunomodulatory therapy in AD patients and indicated PRP as a potential target for future treatments in neuroinflammatory diseases like AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/imunologia , Imunidade Inata/efeitos dos fármacos , Receptores de Peptídeos/administração & dosagem , Receptores de Peptídeos/imunologia , Adulto , Idoso , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Humanos , Imunidade Inata/fisiologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Domínios Proteicos Ricos em Prolina/efeitos dos fármacos , Domínios Proteicos Ricos em Prolina/fisiologia , Receptores de Peptídeos/metabolismo
8.
Biochim Biophys Acta Rev Cancer ; 1869(2): 321-332, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29673969

RESUMO

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.


Assuntos
Caderinas/metabolismo , Movimento Celular , Integrina alfa2beta1/metabolismo , Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação , Caderinas/antagonistas & inibidores , Caderinas/imunologia , Adesão Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/imunologia , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/imunologia , Transdução de Sinais
9.
J Nucl Med ; 59(8): 1234-1242, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29674421

RESUMO

We have developed the 16F12 mouse monoclonal antibody (mAb), which targets the Müllerian-inhibiting substance receptor, type II (MISRII), expressed by ovarian tumors. Here, we assessed in preclinical models the possibility of using radiolabeled 16F12 in a theranostic approach for small-volume ovarian peritoneal carcinomatosis, such as after cytoreductive surgery. Methods: DOTA-, DTPA- or deferoxamine mesylate-conjugated 16F12 mAb was radiolabeled with ß-particle (177Lu) or α-particle (213Bi) emitters for therapeutic use and with 89Zr for PET imaging. On the 13th postxenograft day, mice bearing intraperitoneal MISRII-positive AN3CA endometrial carcinoma cell xenografts were treated by conventional intraperitoneal radioimmunotherapy (IP-RIT) with 10 MBq of 177Lu-16F12 or 12.9 MBq of 213Bi-16F12 or by brief intraperitoneal radioimmunotherapy (BIP-RIT) using 50 MBq of 177Lu-16F12 or 37 MBq of 213Bi-16F12. For BIP-RIT, 30 min after injection of the radiolabeled mAbs, the peritoneal cavity was washed to remove the unbound radioactivity. The biodistribution of 177Lu- and 213Bi-16F12 mAbs was determined and then used for dose assessment. Hematologic toxicity was also monitored. Results: The 16F12 mAb was satisfactorily radiolabeled for both therapy and imaging. IP-RIT with 177Lu-16F12 was slightly more efficient in delaying tumor growth than IP-RIT with 213Bi-16F12. Conversely, 213Bi-16F12 was more efficient than 177Lu-16F12 in BIP-RIT. The biodistribution analysis showed that the tumor-to-blood uptake ratio was significantly higher with BIP-RIT than with IP-RIT for both 213Bi- and 177Lu-16F12. Hematologic toxicity was more pronounced with 177Lu-16F12 than with 213Bi-16F12. SPECT/CT images (after BIP-RIT with 177Lu-16F12) and PET/CT images (after injection of 89Zr-16F12 in the tail vein) showed focal uptake at the tumor site. Conclusion: Radiolabeled 16F12 could represent a new theranostic tool for small-volume ovarian peritoneal carcinomatosis. Specifically, 213Bi-16F12-based BIP-RIT could be proposed to selected patients as an alternative adjuvant treatment immediately after cytoreductive surgery. An anti-MISRII mAb is currently being used in a first-in-human study, thus making radiolabeled anti-MISRII mAbs a realistic theranostic option for the clinic.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/radioterapia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Desferroxamina/química , Feminino , Compostos Heterocíclicos com 1 Anel/química , Humanos , Marcação por Isótopo , Camundongos , Neoplasias Ovarianas/metabolismo , Ácido Pentético/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioquímica , Distribuição Tecidual
10.
Cancer Prev Res (Phila) ; 10(11): 612-624, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29093011

RESUMO

Epithelial ovarian carcinoma (EOC) is the most prevalent form of ovarian cancer in the United States, representing approximately 85% of all cases and causing more deaths than any other gynecologic malignancy. We propose that optimized control of EOC requires the incorporation of a vaccine capable of inducing safe and effective preemptive immunity in cancer-free women. In addition, we hypothesize that ovarian-specific self-proteins that are "retired" from autoimmune-inducing expression levels as ovaries age but are expressed at high levels in emerging EOC may serve as vaccine targets for mediating safe and effective primary immunoprevention. Here, we show that expression of the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in normal tissues is confined exclusively to the human ovary, drops to nonautoimmune inducing levels in postmenopausal ovaries, and is at high levels in approximately 90% of human EOC. We found that AMHR2-ED vaccination significantly inhibits growth of murine EOC and enhances overall survival without inducing oophoritis in aged female mice. The observed inhibition of EOC growth was mediated substantially by induction of AMHR2-ED-specific IgG antibodies that agonize receptor signaling of a Bax/caspase-3-dependent proapoptotic cascade. Our results indicate that AMHR2-ED vaccination may be particularly useful in providing safe and effective preemptive immunity against EOC in women at high genetic or familial risk who have the greatest need for a preventive vaccine and ultimately in cancer-free postmenopausal women who account for 75% of all EOC cases. Cancer Prev Res; 10(11); 612-24. ©2017 AACRSee related editorial by Shoemaker et al., p. 607.


Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/terapia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Idoso , Idoso de 80 Anos ou mais , Vacinas Anticâncer/imunologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Tolerância Imunológica/imunologia , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/patologia , Ooforite/epidemiologia , Ooforite/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ovário/imunologia , Ovário/patologia , Poliendocrinopatias Autoimunes/epidemiologia , Poliendocrinopatias Autoimunes/imunologia , Pós-Menopausa , Proteínas Serina-Treonina Quinases , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinação/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Biol Chem ; 292(21): 8605-8615, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28381555

RESUMO

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.


Assuntos
Proteínas do Capsídeo , Vírus da Doença Hemorrágica de Coelhos , Receptores Imunológicos , Receptores de Peptídeos , Cultura de Vírus/métodos , Motivos de Aminoácidos , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/veterinária , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Chlorocebus aethiops , Cricetinae , Cães , Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/crescimento & desenvolvimento , Vírus da Doença Hemorrágica de Coelhos/imunologia , Humanos , Células Madin Darby de Rim Canino , Coelhos , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Peptídeos/genética , Receptores de Peptídeos/imunologia , Células Vero , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/metabolismo
12.
Oncotarget ; 8(23): 37061-37079, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28427157

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Feminino , Glicosilação , Humanos , Camundongos Nus , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Engenharia de Proteínas
13.
MAbs ; 6(5): 1314-26, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25517316

RESUMO

Ovarian cancer has the highest mortality rate among gynecologic malignancies. The monoclonal antibody 12G4 specifically recognizes the human Müllerian inhibiting substance type II receptor (MISRII) that is strongly expressed in human granulosa cell tumors (GCT) and in the majority of human epithelial ovarian cancers (EOC). To determine whether MISRII represents an attractive target for antibody-based tumor therapy, we first confirmed by immunohistochemistry with 12G4 its expression in all tested GCT samples (4/4) and all, but one, EOC human tissue specimens (13/14). We then demonstrated in vitro the internalization of 12G4 in MISRII(high)COV434 cells after binding to MISRII and its ability to increase the apoptosis rate (FACS, DNA fragmentation) in MISRII(high)COV434 (GCT) and MISRII(medium)NIH-OVCAR-3 (EOC) cells that express different levels of MISRII. A standard (51)Cr release assay showed that 12G4 mediates antibody-dependent cell-meditated cytotoxicity. Finally, in vivo assessment of 12G4 anti-tumor effects showed a significant reduction of tumor growth and an increase of the median survival time in mice xenografted with MISRII(high)COV434 or MISRII(medium)NIH-OVCAR-3 cells and treated with 12G4 in comparison to controls treated with an irrelevant antibody. Altogether, our data indicate that MISRII is a new promising target for the control of ovarian GCTs and EOCs. A humanized version of the 12G4 antibody, named 3C23K, is in development for the targeted therapy of MISRII-positive gynecologic cancers.


Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias Ovarianas/imunologia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Tumor de Células da Granulosa/imunologia , Tumor de Células da Granulosa/metabolismo , Tumor de Células da Granulosa/terapia , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Camundongos Nus , Microscopia de Fluorescência , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapia , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Eur J Immunol ; 44(7): 1956-66, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24771148

RESUMO

Self-tolerance, presumably through lineage-unbiased elimination of self-antigen-specific lymphocytes (CD4(+) T, CD8(+) T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self-tolerance reflects selective elimination of antigen-specific CD4(+) T cells, but preservation of CD8(+) T- and B-cell populations. In mice, antigen-specific CD4(+) T-cell tolerance restricted CD8(+) T- and B-cell responses targeting the endogenous self-antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4(+) T-cell tolerance blocked GUCY2C-specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self-antigen-independent CD4(+) T-cell help. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4(+) T-cell help, revealing the latent functional capacity of GUCY2C-specific CD8(+) T- and B-cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines targeting self-antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4(+) T-cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self-antigens characterized by selective CD4(+) T-cell tolerance and abrogation of such tolerance through self-antigen-independent T-cell help is essential for future immunotherapeutics.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Tolerância Imunológica , Neoplasias/terapia , Animais , Autoantígenos/imunologia , Epitopos de Linfócito T/imunologia , Memória Imunológica , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologia
15.
PLoS One ; 8(11): e79180, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244444

RESUMO

BACKGROUND: Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses. METHODS: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+) and GC-C(-/-) mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-), GC-C(+/+)IL-10(-/-) and GC-C(-/-)IL-10(-/-) mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line. RESULTS: Relative to GC-C(+/+) animals, intraperitoneal lipopolysaccharide injection into GC-C(-/-) mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-)IL-10(-/-) animals was significantly more severe relative to GC-C(+/+)IL-10(-/-) mice. Unlike GC-C(+/+)IL-10(-/-) controls, colon pathology in GC-C(-/-)IL-10(-/-) animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-)IL-10(-/-) mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells. CONCLUSIONS: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.


Assuntos
Colite/imunologia , Tolerância Imunológica , Mucosa Intestinal/imunologia , Mutação , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologia , Transdução de Sinais/imunologia , Animais , Colite/genética , Colite/patologia , Hormônios Gastrointestinais/genética , Hormônios Gastrointestinais/imunologia , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Peptídeos Natriuréticos/genética , Peptídeos Natriuréticos/imunologia , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/genética , Receptores de Peptídeos/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
16.
BMC Gastroenterol ; 13: 135, 2013 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-24004613

RESUMO

BACKGROUND: Guanylate Cyclase C (GC-C) is an apically-oriented transmembrane receptor that is expressed on epithelial cells of the intestine. Activation of GC-C by the endogenous ligands guanylin or uroguanylin elevates intracellular cGMP and is implicated in intestinal ion secretion, cell proliferation, apoptosis, intestinal barrier function, as well as the susceptibility of the intestine to inflammation. Our aim was to determine if GC-C is required for host defense during infection by the murine enteric pathogen Citrobacter rodentium of the family Enterobacteriacea. METHODS: GC-C+/+ control mice or those having GC-C genetically ablated (GC-C-/-) were administered C. rodentium by orogastric gavage and analyzed at multiple time points up to post-infection day 20. Commensal bacteria were characterized in uninfected GC-C+/+ and GC-C-/- mice using 16S rRNA PCR analysis. RESULTS: GC-C-/- mice had an increase in C. rodentium bacterial load in stool relative to GC-C+/+. C. rodentium infection strongly decreased guanylin expression in GC-C+/+ mice and, to an even greater degree, in GC-C-/- animals. Fluorescent tracer studies indicated that mice lacking GC-C, unlike GC-C+/+ animals, had a substantial loss of intestinal barrier function early in the course of infection. Epithelial cell apoptosis was significantly increased in GC-C-/- mice following 10 days of infection and this was associated with increased frequency and numbers of C. rodentium translocation out of the intestine. Infection led to significant liver histopathology in GC-C-/- mice as well as lymphocyte infiltration and elevated cytokine and chemokine expression. Relative to naïve GC-C+/+ mice, the commensal microflora load in uninfected GC-C-/- mice was decreased and bacterial composition was imbalanced and included outgrowth of the Enterobacteriacea family. CONCLUSIONS: This work demonstrates the novel finding that GC-C signaling is an essential component of host defense during murine enteric infection by reducing bacterial load and preventing systemic dissemination of attaching/effacing-lesion forming bacterial pathogens such as C. rodentium.


Assuntos
Colo/imunologia , Infecções por Enterobacteriaceae/imunologia , Mucosa Intestinal/imunologia , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologia , Animais , Apoptose/imunologia , Carga Bacteriana , Translocação Bacteriana/fisiologia , Citrobacter rodentium/fisiologia , Colo/patologia , Infecções por Enterobacteriaceae/genética , Mucosa Intestinal/patologia , Fígado/patologia , Camundongos , Camundongos Knockout , Permeabilidade , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/genética , Receptores de Peptídeos/genética , Transdução de Sinais/imunologia
17.
J Immunol Methods ; 387(1-2): 11-20, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22910001

RESUMO

Prescription of therapeutic antibodies has radically modified the prognosis of some important diseases. However, the very high cost of these new drugs is a problem for public health organizations, which require assessment of the effectiveness of the antibody for each patient before beginning or during the treatment. In vivo immunoimaging is particularly well adapted to meet this demand. However, full-length antibodies are unsuitable for in vivo imaging due to their persistence in the serum and must be engineered in smaller formats to improve their pharmacokinetic properties without modifying their affinity and specificity. The small bivalent antibody fragment called diabody perfectly meets these in vivo imaging requirements. However, obtaining diabodies is laborious, time-consuming and sometimes unsuccessful. Using a diabody derived from a monoclonal antibody (12G4) directed against the human anti-Müllerian hormone receptor, a biomarker of ovarian cancers for which therapeutic antibodies are already undergoing clinical trials, we describe here a new diabody refolding protocol with various reducing conditions. Diabody functionality was checked in vitro and ex vivo with, respectively, a new immunoassay involving the epitopic peptide as a tracer and flow cytometry experiments with cells expressing recombinant anti-Müllerian hormone receptors. Our optimized protocol allows us to find the best refolding conditions for each diabody and to obtain large amounts of functional diabodies.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Neoplasias Ovarianas/imunologia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Sequência de Aminoácidos , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/imunologia , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Epitopos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Corpos de Inclusão/metabolismo , Dados de Sequência Molecular , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Ligação Proteica/imunologia , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
18.
Oncol Rep ; 29(2): 459-63, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23135359

RESUMO

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has recently been identified as one of the vascular endothelial growth factors, and it is considered that the overexpression of EG-VEGF in colon cancer is related to hepatic metastasis. In this study, we report our recent novel findings of the involvement of EG-VEGF in cell invasion of colon cancer cells. Colon cancer cell lines (DLD-1 and HCT116) with high expression of prokineticin receptor (PK-R) 1 and 2 were stimulated with the EG-VEGF protein. Furthermore, Matrigel cell invasion assay was performed to examine the changes in cancer cell invasion. In addition, we investigated the mRNA expression of matrix metalloproteinase (MMP)-2, -7 and -9 in cancer cells. Finally, the EG-VEGF receptor on the colon cancer cell membrane was blocked by anti-PK-R1 and -PK-R2 antibodies to study whether cell invasion ability would be altered. In colon cancer cell lines where the expression of PK-R1 and 2 was confirmed, stimulation with EG-VEGF increased cell invasion a maximum of ~3-5 times. Furthermore, an increase in the mRNA and protein expression of MMP-2, -7 and -9 was observed. We also observed that the cell invasion rate decreased only after exposure to the anti-PK-R2 antibody. The study showed that the EG-VEGF protein may act on MMP-2, -7 and -9 via PK-R2 to strengthen cell invasion ability in colon cancer cell lines.


Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Hormônios Gastrointestinais/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/farmacologia , Anticorpos/farmacologia , Neoplasias do Colo/genética , Hormônios Gastrointestinais/imunologia , Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/imunologia , Receptores de Peptídeos/imunologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/imunologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo
19.
Cancer Immunol Immunother ; 61(5): 713-23, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22057677

RESUMO

Guanylyl cyclase C (GUCY2C) is the index cancer mucosa antigen, an emerging class of immunotherapeutic targets for the prevention of recurrent metastases originating in visceral epithelia. GUCY2C is an autoantigen principally expressed by intestinal epithelium, and universally by primary and metastatic colorectal tumors. Immunization with adenovirus expressing the structurally unique GUCY2C extracellular domain (GUCY2C(ECD); Ad5-GUCY2C) produces prophylactic and therapeutic protection against GUCY2C-expressing colon cancer metastases in mice, without collateral autoimmunity. GUCY2C antitumor efficacy is mediated by a unique immunological mechanism involving lineage-specific induction of antigen-targeted CD8(+) T cells, without CD4(+) T cells or B cells. Here, the unusual lineage specificity of this response was explored by integrating high-throughput peptide screening and bioinformatics, revealing the role for GUCY2C-directed CD8(+) T cells targeting specific epitopes in antitumor efficacy. In BALB/c mice vaccinated with Ad5-GUCY2C, CD8(+) T cells recognize the dominant GUCY2C(254-262) epitope in the context of H-2K(d), driving critical effector functions including interferon gamma secretion, cytolysis ex vivo and in vivo, and antitumor efficacy. The ability of GUCY2C to induce lineage-specific responses targeted to cytotoxic CD8(+) T cells recognizing a single epitope mediating antitumor efficacy without autoimmunity highlights the immediate translational potential of cancer mucosa antigen-based vaccines for preventing metastases of mucosa-derived cancers.


Assuntos
Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Receptores Acoplados a Guanilato Ciclase/imunologia , Receptores de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/imunologia , Animais , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Imunoterapia/métodos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Receptores de Enterotoxina
20.
Biomed Environ Sci ; 24(5): 523-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22108419

RESUMO

OBJECTIVE: To break immune tolerance to prion (PrP) proteins using DNA vaccines. METHODS: Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests. RESULTS: Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response. CONCLUSION: Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.


Assuntos
Tolerância Imunológica , Príons/imunologia , Vacinas de DNA , Animais , Anticorpos/imunologia , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Células HeLa , Humanos , Interferon gama/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Príons/genética , Receptores de Peptídeos/genética , Receptores de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Transfecção , Ubiquitina/genética , Ubiquitina/imunologia
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