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1.
Artigo em Inglês | MEDLINE | ID: mdl-38669775

RESUMO

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.


Assuntos
Anticorpos Monoclonais , Bordetella pertussis , Cromatografia de Afinidade , Fatores de Virulência de Bordetella , Cromatografia de Afinidade/métodos , Animais , Bordetella pertussis/imunologia , Bordetella pertussis/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/imunologia , Camundongos , Fatores de Virulência de Bordetella/imunologia , Fatores de Virulência de Bordetella/química , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/química , Adesinas Bacterianas/isolamento & purificação , Camundongos Endogâmicos BALB C , Ovinos , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/química , Ensaio de Imunoadsorção Enzimática/métodos
2.
Daru ; 31(2): 221-231, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37695454

RESUMO

BACKGROUND: Producing therapeutic proteins can be done quickly and on a large scale through Transient Gene Expression (TGE). Chinese hamster ovary (CHO) cell lines are commonly used to achieve this. Although there are few comparative studies, TGE has been observed in suspension-adapted CHO cells. OBJECTIVES: We tested TGE's effectiveness in DG-44, CHO-S, and ExpiCHO-S cell lines with four transfection reagents. METHODS: A design of experiments (DoE) was followed to optimize transfection using a recombinant monoclonal antibody (mAb) construct. To evaluate the efficacy, flow cytometry and ELISA were used. Feeding strategies and temperature shifts were implemented to enhance transfection effectiveness. The quality of the mAb was assessed through ELISA, SDS-PAGE, and proliferation inhibition assays. RESULTS: We adapted all cell lines to grow in suspension using a serum-free medium. Our findings from flow cytometry and ELISA tests indicate that PEI and Pmax reagents had a higher rate of transfection and mAb production than the ExpiCHO commercial transfection reagent. While DG-44 cells had better transfection efficiency than CHO-S and ExpiCHO-S, there was no significant difference between CHO-S and ExpiCHO-S. Our TGE system was more productive at 32 °C than at 37 °C. In the optimized TGE of Pmax-based transfection in DG-44 at 37 and 32 °C, the production level of mAb was more than half of the amount of the commercial ExpiCHO-S expression system. Still, the number of transfected cells was three times higher, making it more efficient. The purified mAb from all transfected cell lines had similar structural and functional properties under different conditions. CONCLUSION: Our research shows that using Pmax and DG-44 cells in the TGE system is a cost-effective and efficient way to produce humanized monoclonal antibodies. We discovered that this method outperforms the ExpiCHO-S kit.


Assuntos
Anticorpos Monoclonais , Antineoplásicos , Cricetinae , Animais , Cricetulus , Células CHO , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/genética , Proteínas Recombinantes , Expressão Gênica
3.
J Microbiol Methods ; 211: 106786, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37454935

RESUMO

BACKGROUND: Pertussis, or whooping cough, is a highly contagious respiratory disease caused by Bordetella pertussis (BP). Pertactin (PRN) is one of the main immunogenic components of BP and is employed in many commercialized acellular pertussis vaccines (aPVs). Purification of this protein by conventional chromatography methods is challenging and commonly requires multiple laborious processes with low recovery. Using specific monoclonal antibodies (mAbs) for the purification of PRN antigen is expected to yield high purity and recovery of the target molecule. METHODS: Recombinant PRN antigen was used to produce mouse mAbs using hybridoma technology. Structural and functional characteristics of the mAbs were assessed by ELISA, immunoblotting, and flow cytometry. Selected mAbs were employed to purify PRN by affinity chromatography, and the purity and recovery of the purified protein were analyzed by ELISA, SDS-PAGE, and immunoblotting. Moreover, ELISA and flow cytometry techniques were designed using these mAbs to detect PRN in different strains of BP. RESULTS: Five mAbs were produced and selected based on their reactivity with native PRN. Our results demonstrate that purification of PRN by affinity chromatography resulted in a highly pure antigen with 75-85 percent recovery. In addition, ELISA and flow cytometry results indicated that these mAbs could recognize PRN in the bacterial cell walls of different BP strains. CONCLUSION: We successfully produced PRN-specific mAbs and designed an affinity chromatography method to purify PRN antigen with higher purity and recovery than conventional methods. These mAbs could be employed as valuable tools for the detection and purification of PRN for vaccine manufacturing.


Assuntos
Coqueluche , Animais , Camundongos , Coqueluche/diagnóstico , Coqueluche/prevenção & controle , Fatores de Virulência de Bordetella , Bordetella pertussis , Proteínas da Membrana Bacteriana Externa , Vacina contra Coqueluche , Anticorpos Monoclonais , Anticorpos Antibacterianos
4.
J Med Microbiol ; 72(6)2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37387700

RESUMO

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/genética , COVID-19/terapia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Monoclonais , Anticorpos Neutralizantes , Mamíferos
5.
Iran J Immunol ; 20(2): 177-189, 2023 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-37149778

RESUMO

Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.


Assuntos
Anticorpos Monoclonais , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/metabolismo , Biomarcadores Tumorais , Imuno-Histoquímica , Ensaio de Imunoadsorção Enzimática
6.
J Immunother ; 46(4): 121-131, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36939675

RESUMO

Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Camundongos , Animais , Humanos , Camundongos Nus , Trastuzumab , Anticorpos Monoclonais/metabolismo , Receptor ErbB-2 , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Anal Biochem ; 666: 115079, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36754135

RESUMO

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática/métodos , Sensibilidade e Especificidade , Anticorpos Antivirais , Teste para COVID-19
8.
J Cancer Res Clin Oncol ; 149(6): 2437-2450, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35737089

RESUMO

PURPOSE: Several approaches have so far been employed to establish anti-tumor immunity by targeting HER2 protein. Active immunization with recombinant HER2 subdomains has previously been demonstrated to induce potent immune response and tumor growth inhibition. In the present study, we investigated the immunogenicity and tumor inhibitory effect of a fusion protein consisting of human HER2 extracellular subdomain (ECD-DI + II) together with T-helper cell epitopes of Tetanus toxin (p2 and p30). METHODS: BALB/c mice were immunized with two recombinant proteins (DI + II and p2p30-DI + II) emulsified in 4 different adjuvants. Anti-DI + II antibody response, cytokine profile, frequency of splenic CD25+FOXP3+ regulatory T cells (Tregs) and CD8+CD107a+ cytotoxic T lymphocytes (CTLs) were assessed in the immunized mice. To assess the anti-tumor effect, the immunized mice were subcutaneously challenged with HER2-overexpressing tumor cells and the tumor growth was determined. RESULTS: Both recombinant proteins were able to induce comparable levels of ECD-DI + II-specific antibodies. Immunization with p2p30-DI + II resulted in a significant increase in the level of Interferon-gamma (IFN-γ) secretion compared to DI + II protein and significantly higher frequency of CTLs and lower frequency of Tregs. The number of mice that remained tumor-free until day 120 was significantly higher in p2p30-DI + II vaccinated groups. CONCLUSIONS: Our data suggest that the p2p30-DI + II fusion protein together with CpG adjuvant induces more potent anti-tumor immune responses in a mouse tumor model. Accordingly, this formulation might be considered as a potential immunotherapeutic approach in HER2+ cancers.


Assuntos
Genes erbB-2 , Neoplasias , Receptor ErbB-2 , Animais , Humanos , Camundongos , Adjuvantes Imunológicos , Anticorpos , Imunidade , Camundongos Endogâmicos BALB C , Receptor ErbB-2/metabolismo , Proteínas Recombinantes
9.
Asian Pac J Cancer Prev ; 23(9): 2999-3007, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36172662

RESUMO

BACKGROUND: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. METHODS: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. RESULTS: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. CONCLUSION: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.


Assuntos
Neoplasias da Mama , Receptores de Progesterona , Animais , Anticorpos Monoclonais , Neoplasias da Mama/diagnóstico , Receptor alfa de Estrogênio , Estrogênios , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Progesterona , Receptores de Estrogênio , Receptores de Fatores de Crescimento , Receptores de Progesterona/metabolismo , Reprodutibilidade dos Testes
10.
Invest New Drugs ; 39(3): 697-704, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33389387

RESUMO

Immunotherapy of HER2-overexpressing cancers by FDA approved monoclonal antibodies (mAbs) such as trastuzumab and pertuzumab has shown promising results. We have recently produced a novel humanized anti-HER2 mAb, hersintuzumab, which did not sterically inhibit binding of trastuzumab and pertuzumab to HER2, thus recognizing a distinct epitope on subdomain I + II of HER2. In this study, we assessed the in vitro and in vivo anti-tumor activity of this mAb individually and in combination with trastuzumab. Different HER2-overexpressing human cancer cell lines, including SKOV3, NCI-N87 HCC1954 and BT-474 were cultured and binding reactivity of Hersintuzumab to these cell lines was analyzed by flow cytometry. In addition, the inhibitory effect of different concentrations of hersintuzumab, trastuzumab and their combination on tumor cells growth was assessed by XTT assay. For Assessment of tumor growth inhibition in xenograft model, Balb/c athymic nude mice were subcutaneously injected with NCI-N87 and SKOV3 tumor cells and then treated intravenously with these mAbs. Our results showed that hersintuzumab could bind to all HER2-overexpressing cell lines similar to trastuzumab. In vitro experiments showed that both hersintuzumab and trastuzumab individually and in combination inhibited growth of all cell lines with the exception of HCC-1954.Inhibitory effect of the combination of mAbs was significantly higher than that of each mAb alone. Similar results were obtained in the gastric (NCI-N87) and ovarian (SKOV-3) tumor xenograft models. Hersintuzumab in combination with trastuzumab induces synergic anti-tumor effects on HER2-overexpressing cells in vitro and in vivo and is potentially a therapeutic tool for treatment of HER2-overexpressing cancers.


Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos , Neoplasias Ovarianas , Receptor ErbB-2 , Neoplasias Gástricas , Animais , Feminino , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Epitopos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab , Carga Tumoral , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Asian Pac J Cancer Prev ; 21(2): 439-447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32102522

RESUMO

OBJECTIVE: Human epidermal growth factor receptor 3 (HER3) is a unique member of the tyrosine kinase receptors with an inactive kinase domain and is the preferable dimerization partner for HER2 which lead to potent tumorigenic signaling. METHODS: In this study, the expression plasmids coding for the human HER3 subdomains were transfected into CHO-K1 cells. Produced proteins were characterized by ELISA and SDS-PAGE. Rabbits were immunized and produced polyclonal antibodies (pAbs) that were characterized by ELISA, Immunoblotting and flowcytometry and their inhibitory effects were assessed by XTT on BT-474 and JIMT-1 breast cancer cell lines. RESULT: The recombinant subdomains were highly immunogenic in rabbits. The pAbs reacted with the recombinant subdomains as well as commercial HER3 and the native receptor on tumor cell membranes and could significantly inhibit growth of Trastuzumab sensitive (BT-474) and resistant (JIMT-1) breast cancer cell lines in vitro. CONCLUSION: It seems that HER3 extra cellular domains (ECD) induce a strong anti-tumor antibody response and may prove to be potentially useful for immunotherapeutic applications.
.


Assuntos
Anticorpos Antineoplásicos/imunologia , Neoplasias da Mama/imunologia , Domínios Proteicos/imunologia , Receptor ErbB-3/imunologia , Animais , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetulus , Dimerização , Resistencia a Medicamentos Antineoplásicos , Humanos , Imunização Secundária , Imunoglobulina G , Técnicas In Vitro , Camundongos , Plasmídeos , Coelhos , Receptor ErbB-2/metabolismo , Transfecção , Vacinação
12.
Front Immunol ; 11: 600883, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33679691

RESUMO

Overexpression of HER2 has been reported in many types of cancer, making it a perfect candidate for targeted immunotherapy. The combination of two FDA approved monoclonal antibodies (mAbs), trastuzumab and pertuzumab, has more robust anti-tumor activity in patients with HER2-overexpressing breast cancer. We recently produced a new humanized anti-HER2 mAb, hersintuzumab, which recognizes a different epitope than trastuzumab and pertuzumab on HER2. This mAb, in combination with trastuzumab, exhibits more potent anti-tumor activity than each parental mAb alone. Here we have developed a novel bispecific anti-HER2 antibody (BsAb) designated as trasintuzumab, composed of trastuzumab and hersintuzumab, using dual variable domain immunoglobulin (DVD-Ig) technology. Both variable domains of trasintuzumab are fully functional and have similar affinities to the parental mAbs and are also able to bind to natural HER2 on the surface of several HER2-expressing cell lines. Trasintuzumab was found to inhibit the growth of different types of tumor cell lines through suppression of the AKT and ERK signaling pathways as efficiently as the combination of the parental mAbs. It also induced tumor regression as potently as the combination of the two mAbs in nude mice bearing ovarian and gastric cancer xenografts. Our data suggest that trasintuzumab may be a promising BsAb therapeutic candidate for the treatment of HER2-overexpressing cancers.


Assuntos
Anticorpos Biespecíficos , Neoplasias Experimentais/tratamento farmacológico , Receptor ErbB-2 , Células 3T3-L1 , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Células CHO , Cricetulus , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Trastuzumab/imunologia , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Iran J Immunol ; 16(1): 26-42, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30864553

RESUMO

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Macaca fascicularis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transfecção , Trastuzumab/farmacologia
14.
Avicenna J Med Biotechnol ; 11(1): 72-79, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30800246

RESUMO

BACKGROUND: Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer. METHODS: In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA. RESULTS: Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p<0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively. CONCLUSION: These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.

15.
Asian Pac J Cancer Prev ; 19(8): 2255-2262, 2018 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-30139234

RESUMO

Objective: Homo- and heterodimerization of the receptor tyrosine kinase HER2 hyperactivate several downstream signaling pathways, leading to uncontrolled growth and proliferation of tumor cells. Anti-HER2 monoclonal antibodies (mAbs) may induce different effects on HER2 dimerization and signaling. Methods: The effect of two inhibitory (2A8, 1T0) and one stimulatory (1H9) anti-HER2 mAbs either alone or in combination with trastuzumab was investigated on AKT and ERK signaling pathways and HER2 degradation in a human breast cancer cell line (BT-474) by Western blotting. Result: While 1H9 mAb had no significant effect on AKT and ERK signaling pathways, 1T0 and 2A8 mAbs inhibited phosphorylation of both pathways. Combination of 1T0 mAb with trastuzumab resulted in significant synergistic inhibition of both pathways and HER2 degradation, much more potently than the combination of trastuzumab and pertuzumab. Conclusion: Our data indicate that anti-HER2 mAbs may induce different signaling pathways depending on their effect on tumor cell growth and proliferation. The significant inhibition of ERK and AKT phosphorylation by 1T0 alone or particularly in combination with trastuzumab suggests its potential therapeutic application for targeted immunotherapy of HER2 overexpressing malignancies.


Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Receptor ErbB-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
16.
Invest New Drugs ; 36(2): 171-186, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28983766

RESUMO

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/química , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Células CHO , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Mapeamento de Epitopos , Amplificação de Genes , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
17.
Asian Pac J Cancer Prev ; 18(11): 3103-3110, 2017 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-29172286

RESUMO

Background: Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes. Conclusions: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.

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