RESUMO
OBJECTIVE: Human Chorionic Gonadotropin (hCG) plays a crucial role in embryo implantation and in maintenance of pregnancy. An immuno-contraceptive approach involves the use of a recombinant hCGß-LTB vaccine formulated with adjuvant Mycobacterium indicus pranii (MIP), to prevent pregnancy without disturbing ovulation, hormonal profiles, and menstrual cycles in women. The present work in mice was designed to address issues encountered in clinical trials conducted with hCGß-LTB vaccine, with focus on two primary concerns. Firstly, it aimed to determine the optimal vaccine dosage required to induce a high level of anti-hCG antibodies. Secondly, it aimed to assess the safety profile of the vaccine, specifically injection site reactions in the form of nodules, observed in some of the subjects. METHODS AND RESULTS: Studies undertaken indicate that a 2 µg dose of the protein version of the vaccine, administered in mice through the intramuscular route, can induce high anti-hCG titres. Furthermore, administering a booster dose enhances the antibody response. Our findings suggest that the concentration and frequency of administration of the adjuvant MIP can also be reduced without compromising vaccine efficacy. CONCLUSION: The issue of nodule formation at the injection site can be mitigated either by administering the vaccine along with MIP intramuscularly or injecting hCG vaccine and MIP at separate intradermal sites. Thus, protein vaccine administered at a 2µg dose via the intramuscular route addresses both efficacy and safety concerns.
The Phase I/II clinical trials initiated with the recombinant hCG vaccine in women revealed inadequate antibody titres in all subjects, alongside the development of nodules at the injection sites in some participants. Studies were undertaken in mice to propose potential strategies for mitigating injection site reactions and enhancing the antibody response. It was concluded that the optimum dose of the protein version of the vaccine to get high antibody titres, is 2 µg administered intramuscularly while upholding safety standards.
Assuntos
Gonadotropina Coriônica , Vacinas Sintéticas , Animais , Feminino , Camundongos , Vacinas Sintéticas/imunologia , Gonadotropina Coriônica/imunologia , Anticoncepção Imunológica/métodos , Vacinas Anticoncepcionais/imunologia , Formação de Anticorpos/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Adjuvantes Imunológicos , Reação no Local da Injeção , Engenharia Genética , Injeções Intramusculares , Gonadotropina Coriônica Humana Subunidade beta/imunologiaRESUMO
Oral squamous cell carcinoma (OSCC) is an aggressive tumor whose prognosis has little improvement in the last three decades. Various immune-related genes have been suggested as significant roles in the development and progression of malignant cancers. In this study, we acquired and integrated differentially expressed genes of OSCC patients, including immune-related genes and transcription factors (TFs), from The Cancer Genome Atlas (TCGA) database. TF-mediated network was established to exploring the regulatory mechanisms of prognostic immune-related genes. A 7 immune-related genes prognostic model for OSCC was obtained, including CGB8, CTLA4, TNFRSF19, CCL26, NRG1, TPM2 and PLAU, which was further proved to be an independent prognostic indicator after adjusting for other clinical factors. The immune-related genes prognostic index was significantly negatively correlated to the infiltration abundances of B cells (P < 0.05) and CD8+ T cells (P < 0.05). The novel proposed immune-based prognostic model not only provided a promising biomarker and a way to monitor the long-term treatment of OSCC, but also gave a new insight into a potential immunotherapy strategy.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Quimiocina CCL26/genética , Quimiocina CCL26/imunologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Bases de Dados Genéticas , Humanos , Imunidade Celular , Imunoterapia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Genéticos , Neoplasias Bucais/imunologia , Neoplasias Bucais/mortalidade , Neoplasias Bucais/terapia , Neuregulina-1/genética , Neuregulina-1/imunologia , Prognóstico , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Análise de Sobrevida , Fatores de Transcrição/genética , Tropomiosina/genética , Tropomiosina/imunologiaRESUMO
Multiplex immunoassay, or the simultaneous detection of multiple proteins in a single sample, is expected to enable a new level of protein analysis across diverse disciplines, such as medical diagnostics and biomarker discovery. A bead-based assay using graphically encoded hydrogel microparticles synthesized using stop flow lithography has been a promising platform because of its high multiplex capacity and its superior sensitivity and dynamic range compared to the enzyme-linked immunosorbent assay (ELISA). The functionalization of these particles has been dependent on the use of a heterobifunctional linker to conjugate the capture antibodies on the hydrogel. However, the linker chemistry, which is based on linking the primary amine groups of antibodies with acrylate functional groups on the hydrogel monomer, is vulnerable to hydrolysis in aqueous conditions and can potentially damage the antigen binding region of the antibody. In this work, we introduce a new antibody conjugation method that avoids the use of the linker and further enhances the sensitivity of hydrogel microparticle-based immunoassays. Disulfide bonds in antibodies are reduced to liberate free thiols, which can directly bond with the double bonds remaining in the hydrogel after particle synthesis. We characterize the optimal reduction of antibodies for producing the highest detection signal and demonstrate an average two-fold improvement in sensitivity compared to the linker-dependent antibody conjugation method. Lastly, we validate the accuracy and specificity of the multiplex assays with particles conjugated with antibodies using the linker-free method.
Assuntos
Anticorpos/química , Hidrogéis/química , Imunoensaio/instrumentação , Anticorpos/imunologia , Gonadotropina Coriônica Humana Subunidade beta/análise , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Humanos , Imunoensaio/métodos , Limite de Detecção , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.
Assuntos
Adjuvantes Imunológicos/metabolismo , Vacinas Anticâncer/imunologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Neoplasias/prevenção & controle , Animais , Formação de Anticorpos/imunologia , Linhagem Celular , Reações Cruzadas/imunologia , Epitopos/imunologia , Feminino , Humanos , Insetos , Hormônio Luteinizante/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologiaRESUMO
OBJECTIVE: We propose an innovative strategy of nanoparticle-mediated-peptide receptor radionuclide therapy (PRRT) employing PLGA-nanoparticles together with anti-ß-hCG antibodies that can protect kidneys from radiation damage while simultaneously enhancing its tumor targeting and cytotoxic ability for somatostatin receptor (SSR) positive tumors. METHODS: PEG-coated-(177)Lu-DOTATATE-PLGA-nanoparticles (PEG-LuD-NP) were formulated and characterized. In vitro toxicity of these particles was tested on human glioblastoma cell line U87MG over a radiation dose range of 19-78 Gy, using MTT assay and flow cytometry. To further enhance cytotoxicity and test the feasibility of active tumor targeting, apoptosis-inducing anti-ß-hCG monoclonal antibodies were employed in vitro, after confirming expression of ß-hCG on U87MG. In vivo tumor targeting ability of these particles, in comparison to uncoated particles and un-encapsulated (177)Lu-DOTATATE, was assessed by intravenous administration in tumor-induced wistar rats. Rats were first imaged in a gamma camera followed by euthanasia for organ extraction and counting in gamma counter. RESULTS: The particles were spherical in shape with mean diameter of 300 nm. Highest cytotoxicity that could be achieved with PEG-LuD-NP, on radio-resistant U87MG cells, was 35.8 % due to complex cellular response triggered by ionizing radiation. Interestingly, synergistic action of antibodies and PEG-LuD-NP doubled the cytotoxicity (80 %). PEG-LuD-NP showed the highest tumor uptake (4.3 ± 0.46 % ID/g) as compared to (177)Lu-DOTATATE (3.5 ± 0.31 %) and uncoated-(177)Lu-DOTATATE-nanoparticles (3.4 ± 0.35 %) in tumor-inoculated wistar rats (p < 0.001). Renal uptake/retention was decreased 3-4 folds with these particles, resulting in the highest tumor-to-kidney ratio (8.58; p < 0.01) while tumor-to-liver and tumor-to-bone ratios were comparable to un-encapsulated-drug. CONCLUSION: Nanocarrier-mediated-PRRT is an effective way of targeting SSR positive tumors for enhanced cytoxicity and reduced renal radiation dose associated with conventional PRRT. To our knowledge of literature, this is the first study to establish in vitro and in vivo efficacy profile of nanoparticles in PRRT providing a stepping-stone for undergoing and future research endeavors in the direction of abating associated radiation concerns of radionuclide therapy and may offer a paradigm shift in PRRT strategy.
Assuntos
Glioblastoma/patologia , Terapia de Alvo Molecular , Nanopartículas/química , Octreotida/análogos & derivados , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Receptores de Somatostatina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Estudos de Viabilidade , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , Ácido Láctico/química , Masculino , Octreotida/química , Octreotida/metabolismo , Octreotida/farmacologia , Octreotida/uso terapêutico , Compostos Organometálicos/metabolismo , Compostos Organometálicos/uso terapêutico , Polietilenoglicóis/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RatosRESUMO
To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and ß-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, ß-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.
Assuntos
Vacinas Anticâncer/imunologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Melanoma Experimental/terapia , Neovascularização Patológica/prevenção & controle , Neoplasias Cutâneas/terapia , Vacinas de DNA/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Gonadotropina Coriônica Humana Subunidade beta/antagonistas & inibidores , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Feminino , Expressão Gênica , Células HEK293 , Humanos , Imunização , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/genética , Interleucina-12/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/química , Plasmídeos/metabolismo , Replicon , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Survivina , Resultado do Tratamento , Vacinas Combinadas , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
PROBLEM: Necessity to elicit antibody response above the protective threshold titres by sexually active women immunized to prevent pregnancy. METHOD OF STUDY: Recombinant hCGß-LTB vaccine expressed as both DNA and protein. Balb C mice employed for testing immunogenicity. RESULTS: Necessity to give three primary injections of the vaccine to elicit proper antibody response. Immunization twice with DNA form of the vaccine at fortnightly interval followed by the protein elicits a distinctly higher antibody response than proteinic vaccine alone. Antibodies generated are bio-effective against hCG. CONCLUSION: Immunization with the DNA form of the recombinant hCGß-LTB vaccine twice at fortnightly interval followed by the proteinic form of the vaccine induces distinctly higher antibody response.
Assuntos
Toxinas Bacterianas/imunologia , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Anticoncepção Imunológica/métodos , DNA/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas Anticoncepcionais/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos/imunologia , Formação de Anticorpos/imunologia , Toxinas Bacterianas/genética , Gonadotropina Coriônica Humana Subunidade beta/genética , DNA/administração & dosagem , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Proteínas Recombinantes de Fusão/genética , VacinaçãoRESUMO
Previous studies have documented that selective delivery of protein antigens to cells expressing mannose receptor (MR) can lead to enhanced immune responses. We postulated that agents that influenced the MR expression level, and the activation and migration status of MR-expressing antigen presenting cells, would modulate immune responses to MR-targeted vaccines. To address this question, we investigated the effect of clinically used adjuvants in human MR transgenic (hMR-Tg) mice immunized with an MR-targeting cancer vaccine composed of the human anti-MR monoclonal antibody B11 fused with the oncofetal protein, human chorionic gonadotropin beta chain (hCGß), and referred to as B11-hCGß. We found that humoral responses to low doses of B11-hCGß could be enhanced by prior administration of GM-CSF, which upregulated MR expression in vivo. However, co-administration of the Toll-like receptor (TLR) agonists, poly-ICLC and/or CpG with B11-hCGß was required to elicit Th1 immunity, as measured by antigen-specific T-cell production of IFN-γ. The TLR agonists were shown to increase the number of vaccine-containing cells in the draining lymph nodes of immunized hMR-Tg mice. In particular, with B11-hCGß and poly-ICLC, a dramatic increase in vaccine-positive cells was observed in the T-cell areas of the lymph nodes, compared to the vaccine alone or combined with GM-CSF. Importantly, the absence of the TLR agonists during the priming immunization led to antigen-specific tolerance. Therefore, this study provides insight into the mechanisms by which adjuvants can augment immune responses to B11-hCGß and have implications for the rationale design of clinical studies combining MR-targeted vaccination with TLR agonists.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Vacinas Anticâncer/genética , Carboximetilcelulose Sódica/análogos & derivados , Lectinas Tipo C/genética , Lectinas de Ligação a Manose/genética , Oligodesoxirribonucleotídeos/farmacologia , Poli I-C/farmacologia , Polilisina/análogos & derivados , Receptores de Superfície Celular/genética , Linfócitos T/efeitos dos fármacos , Receptores Toll-Like/agonistas , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Carboximetilcelulose Sódica/farmacologia , Gonadotropina Coriônica Humana Subunidade beta/administração & dosagem , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunidade Celular/efeitos dos fármacos , Interferon gama/genética , Interferon gama/imunologia , Lectinas Tipo C/imunologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Contagem de Linfócitos , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Transgênicos , Polilisina/farmacologia , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
We compared how measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic gonadotropin (fß-hCG) in maternal blood are influenced by different methods for blood collection, sample matrix, and immunoassay platform. Serum and dried blood spots (DBS) were obtained by venipuncture and by finger prick of 19 pregnant women. PAPP-A and fß-hCG from serum and from DBS were measured by conventional indirect immunoassay on an AutoDELFIA platform and by antibody microarray. We compared methods based on the recoveries for both markers as well as marker levels correlations across samples. All method comparisons showed high correlations for both marker concentrations. Recovery levels of PAPP-A from DBS were 30% lower, while those of fß-hCG from DBS were 50% higher compared to conventional venipuncture serum. The recoveries were not affected by blood collection or immunoassay method. The high correlation coefficients for both markers indicate that DBS from finger prick can be used reliably in a prenatal screening setting, as a less costly and minimally invasive alternative for venipuncture serum, with great logistical advantages. Additionally, the use of antibody arrays will allow for extending the number of first trimester screening markers on maternal and fetal health.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Teste em Amostras de Sangue Seco/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/normas , Gonadotropina Coriônica Humana Subunidade beta/sangue , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Teste em Amostras de Sangue Seco/normas , Feminino , Humanos , Imunoensaio/métodos , Gravidez , Proteína Plasmática A Associada à Gravidez/análise , Proteína Plasmática A Associada à Gravidez/imunologia , Diagnóstico Pré-Natal/normasRESUMO
Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked ß subunit of hCG (hCGßn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGßn44/45 and hCGß47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGß peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGß, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGß-mAbs cannot discern between hCGßn44/45, hCGßn47/48, or intact hCGß as all 15 mAbs recognizing hCGß also recognized both nicked variants irrespective of which of the three major hCGß antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGßCTP. LC-MS/MS confirmed that their epitopes were not located on hCGß peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGßn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.
Assuntos
Anticorpos Monoclonais/imunologia , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Cromatografia Líquida/métodos , Epitopos/genética , Feminino , Humanos , Espectrometria de Massas/métodos , Gravidez , Valores de Referência , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: To observe the influence of Fe3O4-dextran-anti-ß-human chorionic gonadotropin (HCG) carrying heparanase (Hpa) antisense oligodeoxynucleotide (ASODN), via the invasion, proliferation, and Hpa expression of JEG-3 cell lines and inhibitory effect of transplanted choriocarcinoma tumor growth. METHODS: The different abilities of invasion and proliferation between transfected JEG-3 and untransfected JEG-3 were measured by Matrigel invasion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro. The effect of Hpa ASODN transfection on the expression of Hpa mRNA and protein was measured by reverse-transcription polymerase chain reaction and Western blot. The transplanted choriocarcinoma tumors were taken out to calculate the inhibitory effect on tumor growth of Hpa ASODN. RESULTS: IN THIS STUDY, WE FOUND THAT: (1) the invasive ability of JEG-3 cells was inhibited sufficiently (P < 0.05) after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN; (2) after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN at 48 and 72 hours, the proliferative ability of JEG-3 cells was inhibited sufficiently (P < 0.05); (3) the expression of Hpa mRNA and protein in JEG-3 cells was inhibited efficiently after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN (P < 0.05); and (4) Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN had an inhibitory effect on the transplanted choriocarcinoma tumor growth (P < 0.05) and was harmless on nude mice. CONCLUSION: Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN weakened the invasive and proliferative ability of choriocarcinoma, with a significant inhibitory effect on the transplanted choriocarcinoma tumor. Therefore, Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN is an effective gene therapy, and Fe3O4-dextran-anti-ßHCG nanoparticles are a harmless and effective gene vector.
Assuntos
Anticorpos Monoclonais/metabolismo , Coriocarcinoma/tratamento farmacológico , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Glucuronidase/genética , Nanopartículas de Magnetita/química , Oligodesoxirribonucleotídeos Antissenso/genética , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Dextranos , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Glucuronidase/análise , Glucuronidase/metabolismo , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To obtain ß-chain human chorionic gonadotropin (ß-hCG) fusion protein (ß-hCG/GST) and identify its antigenicity. METHODS: The full-length gene of ß-hCG was amplified by PCR. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-ß-hCG, and then it was transformed into BL21 (DE3) for ß-hCG/GST fusion protein expression under IPTG induction. After SDS-PAGE assay, the fusion protein was purified by affinity chromatography and identified by Western blotting. The antigenicity of the purified fusion protein was characterized by ELISA. RESULTS: The ß-hCG gene we obtained had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector pET-42a-ß-hCG was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated that the purified ß-hCG fusion protein had satisfactory antigenicity. CONCLUSION: The purified ß-hCG/GST fusion protein with satisfactory antigenicity has been obtained, which will facilitate further study on active anti-tumor immunotherapy targeting ß-hCG.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes de Fusão , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Humanos , Plasmídeos/genética , Reação em Cadeia da PolimeraseRESUMO
The ß-subunit of human chorionic gonadotropin (ß-hCG) is ectopically expressed in various types of cancer and has been utilized as an antigenic target in anti-cancer vaccines. In view of the low immunogenicity of this self-peptide, we designed a method based on the isocaudamer technique to generate 14 tandem repeats of the 10-residue sequence X of ß-hCG (109-118). These tandemly repeated copies were then combined with ß-hCG C-terminal 37 peptides (CTP37) and finally fused to mycobacterial heat-shock protein 65 (HSP65) to construct a fusion protein HSP65-X14-ßhCGCTP37 as an immunogen. In this study, BALB/c female mice were immunized via subcutaneous injection of the designed protein. Humoral immune and cellular immune responses were effectively elicited. A high titer of anti-ß-hCG antibody was detected in immunized mice sera by enzyme-linked immunosorbent assay and verified by Western blot analysis. The fusion protein, HSP65-X14-ß-hCGCTP37, effectively inhibited the growth of Ehrlich ascites carcinoma in mice. These results suggest that HSP65-X14-ßhCGCTP37 may be an effective tumor vaccine, and the use of multiple tandem repeats of a certain epitope is an effective method to overcome the low immunogenicity of self-peptide antigens.
Assuntos
Proteínas de Bactérias/genética , Vacinas Anticâncer/imunologia , Chaperonina 60/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Sequência de Bases , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Gonadotropina Coriônica Humana Subunidade beta/genética , Feminino , Ordem dos Genes , Humanos , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/genética , VacinaçãoRESUMO
The present study aimed to explore the possibility of developing a immuno-imaging/therapeutic agent for hCG-expressing tumors by using antibodies raised against them. Three human cancer cell lines were selected:lung adenocarcinoma (A549), glioblastoma (U87MG) and breast cancer (MCF7). Anti-ß-hCG monoclonal antibody, obtained from ascitic fluid, was purified by affinity chromatography followed by characterization and titration. Ectopic expression of hCG on these cell lines was tested by flow cytometry and in-vitro cytotoxicity with antibodies was tested by MTT assay on the cell line with the highest percentage binding. For positive and negative controls, immortalized trophoblast cells (SW71) and peripheral blood monocytes were used. Antibody was then radiolabeled with lutetium-177 ((177)Lu) and in vivo biodistribution studies were conducted in murine tumor model. Antibodies could be purified to homogeneity with a concentration 28mg/mL. Percentage receptor expression on A549, U87MG and MCF7 cells was 95%, 66% and 55% respectively. About 90% of A549 cells could be killed with antibody at 72h post-treatment. No cytotoxicity was observed on SW71 despite a high binding percentage (96%). Antibodies were radiolabeled with high efficiency (â¼98%). In-vivo studies using radiolabeled antibodies showed hepato-biliary excretion route and significant uptake in A549 tumor. In conclusion, among the 3 cancer cell lines, lung adenocarcinoma significantly expresses ß-hCG and shows dose dependent cytotoxicity with anti-ß-hCG antibody. Radiolabeling of this antibody can aid in imaging and also has the potential of enhancing its therapeutic potential. This study provides a platform for further studies for targeted radio-immuno imaging and subsequent therapy of hCG-expressing cancers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Terapia de Alvo Molecular/métodos , Radioimunodetecção/métodos , Radioimunoterapia/métodos , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/radioterapia , Adenocarcinoma de Pulmão , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/radioterapia , Masculino , Peso Molecular , Ratos , Ratos WistarRESUMO
The objective of this study was to develop a magnetic particle-linked monoclonal antibody to hCG ß for immunosorbent assay of human chorionic gonadotropin (hCG) with improved detection sensitivity. Monoclonal antibody against hCG ß was found to be optimally cross-linked to the superparamagnetic particles (SPIO) using EDC and NHS as cross-linking reagents. This superparamagnetic particle-linked monoclonal antibody was able to concentrate hCG from a tested solution for further ELISA assay using horse radish peroxidase-labeled monoclonal antibody against hCG ß. This hybrid technique had greatly decreased the detection limit to 0.1 mIU/mL, making an early detection of pregnancy possible. With an improved sensitivity and simple operation, the magnetic particle-linked anti hCG ß antibody for immunoassay of human chorionic gonadotropin (hCG) has a great potential to supersede the traditional ELISA for pregnancy diagnosis.
Assuntos
Anticorpos Monoclonais/imunologia , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Imunoensaio/métodos , Nanopartículas de Magnetita/química , Testes de Gravidez/métodos , Anticorpos Monoclonais/química , Gonadotropina Coriônica Humana Subunidade beta/análise , Feminino , Humanos , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
PURPOSE: The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self-antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. EXPERIMENTAL DESIGN: Two phase I studies were conducted with CDX-1307, a vaccine composed of human chorionic gonadotropin beta-chain (hCG-ß) fused to an MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose escalation of single-agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and the Toll-like receptor (TLR) 3 agonist polyinosinic-polycytidylic acid (poly-ICLC) and TLR7/8 agonist resiquimod to activate the APC. RESULTS: CDX-1307 induced consistent humoral and T-cell responses to hCG-ß when coadministered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and nonelevated levels of serum hCG-ß. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. CONCLUSIONS: APC targeting and activation induce adaptive immunity against poorly immunogenic self-antigens which has implications for enhancing the efficacy of cancer immunotherapy.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Autoantígenos/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Receptores Toll-Like/agonistas , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/metabolismo , Vacinas Anticâncer/farmacocinética , Vacinas Anticâncer/toxicidade , Linhagem Celular Tumoral , Gonadotropina Coriônica Humana Subunidade beta/sangue , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Feminino , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Masculino , Estadiamento de Neoplasias , Neoplasias/patologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/toxicidade , Pele/imunologia , Pele/metabolismo , Pele/patologia , Receptores Toll-Like/metabolismo , Resultado do TratamentoRESUMO
Cancer vaccines have demonstrated clinical benefit, however greater efficacy could be achieved by enhancing their immunogenicity. Owing to cancer vaccines depending on uptake and cross-presentation of tumor antigens by antigen-presenting cells (APCs), we hypothesized that greater immunogenicity would accompany strategies that direct antigen to APC-expressed mannose receptors, initiating a pathway increasing class I and II presentation to T cells. CDX-1307 consists of a human monoclonal antibody targeting the mannose receptor, fused to the human chorionic gonadotropin-ß chain (hCG-ß), a tumor antigen frequently expressed by epithelial cancers including bladder cancer. In Phase I studies of cancer patients, CDX-1307 was well tolerated and induced significant hCG-ß-specific cellular and humoral immune responses when co-administered with GM-CSF and the Toll-like receptor agonists resiquimod and poly-ICLC. An ongoing Phase II trial evaluates CDX-1307 in patients with newly diagnosed, resectable, hCG-ß-expressing bladder cancer, where low tumor burden and early intervention may provide greater potential for benefit.
Assuntos
Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/sangue , Vacinas Anticâncer/efeitos adversos , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Humanos , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Músculos/patologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Resultado do Tratamento , Bexiga Urinária/patologiaRESUMO
Human chorionic gonadotropin (hCG) is synthesized soon after fertilization and is essential for embryonic implantation. A vaccine targeting hCG would be an ideal choice for immuno-contraception; an anti-hCG vaccine developed by Talwar et al., has previously undergone Phase II efficacy trials, providing proof of principle. These trials established the threshold levels of bio-neutralizing anti-hCG antibody titers required to prevent pregnancy; however, these titers (>50 ng/ml) were achieved in only 80% of immunized women. In this communication, we report a novel recombinant anti-hCG vaccine which demonstrates improved immunogenicity. hCGß was genetically fused at C-terminal to the B-subunit of E. coli heat-labile enterotoxin. The recombinant fusion protein (hCGß-LTB) was expressed in Pichia pastoris and, upon adsorption on Alhydrogel along with Mycobacterium indicus pranii (MIP) as an immuno-modulator, evoked a very high anti-hCG immune response in 100% of immunized BALB/c mice. This recombinant vaccine is expected to reduce cost as well as facilitate production of a molecularly consistent conjugate on a large scale.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/imunologia , Vacinas Anticoncepcionais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/isolamento & purificação , Clonagem Molecular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/imunologiaRESUMO
To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the ß-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ~1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the ß5, ß9 and ß8 BCEs of ß-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional ß5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of ß-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/imunologia , Epitopos de Linfócito B/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Western Blotting , Gonadotropina Coriônica Humana Subunidade beta/química , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitopos de Linfócito B/química , Epitopos de Linfócito B/metabolismo , Escherichia coli , Feminino , Camundongos , Tamanho do Órgão , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Estereoisomerismo , Útero/metabolismo , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/metabolismo , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismoRESUMO
Human chorionic gonadotropin (hCG) is a membrane-associated protein highly expressed in several types of human cancer cells. The expression in the cancer cells indicates that hCG may be a potential target molecule for cancer immunotherapy. The objective of this study was to develop a novel immunogenic molecule, which can efficiently induce the neutralizing antibody against hCG and which is also suitable for mass production. The immunogenicity of the recombinant single chain chimeric protein of hCGß-oLHα expressed by yeast was examined. Additionally, the inhibitory effects of the anti-hCGß-oLHα antibody on the growth of hCG-positive cancer cells were determined. It was found that hCGß-oLHα yielded high titers of anti-hCG rabbit antibody that could effectively neutralize the bioactivity of hCG. The rabbit anti-hCGß-oLHα IgG inhibited the proliferation of hCG-expressing human colorectal cancer cells (LS-174, HCT-116, HCT-15 and KM-12) in a dose-dependent manner. Furthermore, an intact anti-tumor vaccine was prepared by conjugating hCGß-oLHα with tetanus toxoid (TT) and this was used to immunize Balb/c mice bearing hCG-expressing SP2/0 tumor cells. The progression of tumors in these immunized mice was remarkably inhibited. These results suggest that hCGß-oLHα is a new promising immunogenic molecule for the development of an anti-hCG-based cancer vaccine.