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Virus infection induces B cells with a wide variety of B cell receptor (BCR) repertoires. Patterns of induced BCR repertoires are different in individuals, while the underlying mechanism causing this difference remains largely unclear. In particular, the impact of germ line BCR immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent antibody induction associated with a germ line BCR Ig gene polymorphism. B404-class antibodies, which were previously reported as potent anti-simian immunodeficiency virus (SIV) neutralizing antibodies using the germ line VH3.33 gene-derived Ig heavy chain, were induced in five of 10 rhesus macaques after SIVsmH635FC infection. Investigation of VH3.33 genes in B404-class antibody inducers (n = 5) and non-inducers (n = 5) revealed association of B404-class antibody induction with a germ line VH3.33 polymorphism. Analysis of reconstructed antibodies indicated that the VH3.33 residue 38 is the determinant for B404-class antibody induction. B404-class antibodies were induced in all the macaques possessing the B404-associated VH3.33 allele, even under undetectable viremia. Our results show that a single nucleotide polymorphism in germ line VH genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line VH-gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.IMPORTANCE Vaccines against a wide variety of infectious diseases have been developed mostly to induce antibodies targeting pathogens. However, small but significant percentage of people fail to mount potent antibody responses after vaccination, while the underlying mechanism of host failure in antibody induction remains largely unclear. In particular, the impact of germ line B cell receptor (BCR)/antibody immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent anti-simian immunodeficiency virus neutralizing antibody induction associated with a germ line BCR/antibody Ig gene polymorphism in rhesus macaques. Our results demonstrate that a single nucleotide polymorphism in germ line Ig genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line BCR/antibody Ig gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.
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In current study, we report the direct interaction force comparison for a synthesized peptide probe to actin and cofilin protein to actin using atomic force microscopy. The peptide probe was synthesized following the actin binding module of cofilin protein. Thus, the functionality of the peptide probe was similar with that of the cofilin protein. The difference between the peptide probe and cofilin protein was the molecular size. The small peptide probe enables highly dense surface modification, thus produces different interaction force curves compared with that of original cofilin protein to actin. The results showed the peptide probe was possible to measure many numbers of the interaction force though the measurement of single molecular order interaction force was a weak point. These imply that the peptide probe has a merit when that was applied surface related applications such as protein sensor and protein-protein interaction separation.
Assuntos
Fatores de Despolimerização de Actina/química , Actinas/química , Peptídeos/química , Microscopia de Fluorescência , Sondas MolecularesRESUMO
This paper presents a direct interaction force measurement between histidine molecules using AFM force-distance curve measurement. AFM force-distance curves between the histidine-modified cantilever and substrate in the different conditions with or without intercalating Cu2+ ion were measured and interpreted via Gaussian curve fitting analyses. The adhesion force between histidine molecules was shown to be 110 pN under the presence of Cu2+. The result was compareable to the measured adhesion force about 0 pN, which was measured by the removal of Cu2+ ion with the addition of EDTA. The result indicated the direct histidine-histidie interaction was difficult without the role of the bridigible ionic component. From the results, the possibility of direct measurement on chemical affinities between biomolecules was suggested by using AFM force-distance curve analyses. Especially, the current approach showed the possible affinity measurement techniques that elucidate the role of bridge ions.
Assuntos
Cobre/química , Histidina/química , Microscopia de Força Atômica/métodos , Mapeamento de Interação de Proteínas/métodos , Adesividade , Sítios de Ligação , Íons , Ligação Proteica , Estresse MecânicoRESUMO
According to guidelines, carbon-ion beam therapy is considered to carry a high safety risk for patients with cardiac implantable electronic devices (CIEDs), although the actual impacts remain unclear. In this study, we investigated the safety of carbon-ion beam therapy in patients with CIEDs. Patients with CIEDs who underwent carbon-ion therapy at Gunma University Heavy Ion Medical Center between June 2010 and December 2019 were identified and investigated for abnormalities in the operation of their CIEDs, such as oversensing and resetting during irradiation, and abnormalities in operation after treatment. In addition, the risk of irradiation from carbon-ion beam therapy was evaluated by model simulations. Twenty patients (22 sites) with CIEDs were identified, 19 with pacemakers and one with an implantable cardioverter-defibrillator (ICD). Treatments were completed without any problems, except for one case in which the treatment was discontinued because of worsening of the primary disease. Monte Carlo simulation indicated that the carbon beam irradiation produced neutrons at a constant and high level in the irradiation field. Nevertheless, with the distances between the CIEDs and the irradiation fields in the analyzed cases, the quantity of neutrons at the CIEDs was lower than that within the irradiation. Although carbon-ion beam therapy can be safely administered to patients with CIEDs, it is advisable to perform the therapy with sufficient preparation and backup devices because of the risks involved.
Assuntos
Desfibriladores Implantáveis , Radioterapia com Íons Pesados , Marca-Passo Artificial , Carbono/uso terapêutico , Desfibriladores Implantáveis/efeitos adversos , Eletrônica , HumanosRESUMO
The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFR-expressing canine fibroblastic tumors.
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Antineoplásicos , Neoplasias , Animais , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Cricetinae , Cricetulus , Cães , Receptores ErbB , Xenoenxertos , Humanos , CamundongosRESUMO
The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.
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Anticorpos Monoclonais , Neoplasias Mamárias Animais , Animais , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cães , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Imunoglobulina G , Neoplasias Mamárias Animais/tratamento farmacológico , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore, 134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry. In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cell line, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly suppressed the development of D-17 compared with the results in response to control mouse IgG. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could be valuable as part of an antibody treatment regimen for them.
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Anticorpos Monoclonais , Osteossarcoma , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Cricetinae , Cães , Xenoenxertos , Humanos , Camundongos , Osteossarcoma/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays an important role in normal epidermal cell physiology. EGFR is overexpressed in cancer cells and has a number of mutations that implicate tumor malignancy, development, and poor patient prognosis; thus, EGFR is an attractive target for cancer therapy. At present, anti-EGFR monoclonal antibodies (mAbs) have been approved and are used for treating patients with a variety of EGFR-expressing cancers. Epitope mapping is important in identifying the therapeutic mechanism of anti-EGFR mAbs; however, the development of epitope mapping techniques lags behind the development of antimolecular target mAbs, including anti-EGFR mAbs. Hence, in this study, a novel epitope mapping method, RIEDL insertion for epitope mapping (REMAP) method, was developed. The results of this study demonstrated that the critical epitope of anti-EGFR mAb EMab-134 is Gly378, Asp379, Ser380, Phe381, Thr382, His383, Thr384, Pro385, and Pro386 of EGFR. The REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.
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Anticorpos Monoclonais , Neoplasias , Mapeamento de Epitopos , Epitopos , Receptores ErbB/genética , HumanosRESUMO
The development of specific antibodies is essential to understand a wide variety of biological phenomena and pathophysiological analyses. Podoplanin (PDPN), a type I transmembrane glycoprotein, is known as a diagnostic marker. Anti-PDPN monoclonal antibodies (mAbs) against many species, such as human, mouse, rat, rabbit, dog, bovine, cat, tiger, horse, pig, goat, alpaca, Tasmanian devil, bear, whale, and sheep, have been established in recent studies. However, sensitive and specific mAbs against elephant PDPN (elePDPN) have not been established. Thus, this study established a novel mAb against African savanna elephant (Loxodonta africana) PDPN using the Cell-Based Immunization and Screening method. elePDPN-overexpressed Chinese hamster ovary-K1 (CHO/elePDPN) cells were immunized, and mAbs were screened against elePDPN using flow cytometry. One of the mAbs, PMab-265 (IgM, κ), specifically detected CHO/elePDPN cells by flow cytometry. These findings suggested the potential usefulness of PMab-265 for the functional analyses of elePDPN.
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Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Elefantes/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Cricetulus , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Podócitos/imunologia , RatosRESUMO
TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.
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CC chemokine receptor 8 (CCR8) belongs to the class A of G protein-coupled receptor. It is highly expressed on Treg and T helper 2 (TH2) cells recruited to the inflammation site and is implicated in allergy and asthma. Recently, CCR8+Treg cells have been suggested to be a master regulator in the immunosuppressive tumor microenvironment; therefore, developing sensitive monoclonal antibodies (mAbs) for CCR8 has been desired. This study established a specific and sensitive mAb for mouse CCR8 (mCCR8), which is useful for flow cytometry by using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCCR8 mAb, C8Mab-2 (rat IgG2b, kappa), reacted with mCCR8-overexpressed Chinese hamster ovary-K1 (CHO/mCCR8) cells and P388 (mouse lymphoid neoplasma) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR8 by flow cytometry. C8Mab-2, which was established by the CBIS method, could be useful for elucidating the mCCR8-related biological response by flow cytometry.
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Anticorpos Monoclonais/imunologia , Receptores CCR8/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Camundongos , Ratos , Receptores CCR8/antagonistas & inibidoresRESUMO
CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled receptors and is present in high levels in eosinophils, basophils, and airway epithelial cells. CCR3 is considered the therapeutic target for human immunodeficiency virus (HIV) infections and allergic diseases; therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR3 has been desired. This study aimed to establish a specific and sensitive mAb against mouse CCR3 (mCCR3) useful for flow cytometry analysis by employing the Cell-Based Immunization and Screening (CBIS) method. The generated anti-mCCR3 mAb, C3Mab-2 (rat IgG2b, kappa), was found to react with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells, according to flow cytometric analysis. Also, it reacted with P388 (mouse lymphoid neoplasm) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR3. Taken together, C3Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.
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Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/farmacologia , Infecções por HIV/imunologia , Receptores CCR3/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Células CHO , Cricetulus , Citometria de Fluxo , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Macrófagos/imunologia , Camundongos , Receptores CCR3/antagonistas & inibidoresRESUMO
CC chemokine receptor 9 (CCR9) belongs to the beta chemokine receptor family and is mainly distributed on the surface of immature T lymphocytes and enterocytes. This receptor is highly expressed in rheumatoid arthritis, colitis, type 2 diabetes, and various tumors. Therefore, more sensitive monoclonal antibodies (mAbs) need to be developed to predict the prognosis of many high CCR9 expression diseases. Because CCR9 is a structurally unstable G protein-coupled receptor, it has been difficult to develop anti-CCR9 mAbs using the traditional method. This study developed anti-human CCR9 (hCCR9) mAbs for flow cytometry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with hCCR9-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hCCR9), and hybridomas showing strong signals from CHO/hCCR9 and no signals from CHO-K1 cells were selected by flow cytometry. We established an anti-hCCR9 mAb, C9Mab-1 (IgG1, kappa), which detected hCCR9 in MOLT-4 leukemia T lymphoblast cells and CHO/hCCR9 cells by flow cytometry. Our study showed that an anti-hCCR9 mAb was developed more rapidly by the CBIS method than the previous method.
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Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Receptores CCR/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/farmacologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Células CHO , Colite/imunologia , Colite/terapia , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/terapia , Enterócitos/imunologia , Epitopos/imunologia , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Camundongos , Receptores CCR/antagonistas & inibidores , Linfócitos T/efeitos dos fármacosRESUMO
Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN (seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63-82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alanine-substituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope might contribute to the pathophysiological investigations of seaPDPN.
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Leões-Marinhos , Alanina , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Glicoproteínas de Membrana/genética , Camundongos , MutagêneseRESUMO
Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1 (PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2, resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than their single use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new anti-human TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG1, kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (KD) of TgMab-2 for CHO/hTIGIT cells were determined to be 3.5 × 10-9 M. These results suggest that TgMab-2, which was developed by CBIS method, is useful for analyzing the function of hTIGIT by flow cytometry.
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Anticorpos Monoclonais/imunologia , Inibidores de Checkpoint Imunológico/imunologia , Neoplasias/terapia , Receptores Imunológicos/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Células CHO , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Cricetinae , Cricetulus , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive antiTROP2 monoclonal antibody (clone TrMab6; mouse IgG2b, κ) was developed using a CellBased Immunization and Screening (CBIS) method. TrMab6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab6 possesses in vitro antibodydependent cellular cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2overexpressed CHOK1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDAMB231, and MDAMB468. In vitro experiments revealed that TrMab6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHOK1 and MCF7/TROP2knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 xenografts revealed that TrMab6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHOK1 and MCF7/TROP2knockout xenografts. The findings suggest that TrMab6 is a promising treatment option for TROP2expressing breast cancers.
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Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células CHO , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Feminino , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.
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Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Leões-Marinhos/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetinae , Cricetulus , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/imunologia , Leões-Marinhos/genéticaRESUMO
Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed in various cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from a variety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, we evaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistry use, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated intense PMab-241 staining within Bowman's capsules and glomeruli of the ferret kidney, and lymphatic endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of PDPN in ferret tissues.
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Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Furões/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Cricetulus , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Podócitos/imunologia , RatosRESUMO
Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a humanized anti-HER2 monoclonal antibody (mAb), has been shown to provide significant survival benefits in HER2-overexpressing breast cancer and gastric cancer patients. Previously, an anti-HER2 mAb, H2Mab-41 (IgG2b, kappa), was developed in our laboratory and its antitumor activity was demonstrated in mouse xenograft models of human colon cancer. The present study aimed to investigate the ability of H2Mab-41 to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dog HER2 (dHER2)-overexpressed cell lines, and thus exert its antitumor activity against dHER2-overexpressed tumors in vivo. Flow cytometry results demonstrated the cross-reactivity of H2Mab-41 with dHER2. Further evaluation of interaction between H2Mab-41 and dHER2-overexpressed CHO-K1 (CHO/dHER2) cells indicated moderate binding affinity of H2Mab-41 toward dHER2, with a dissociation constant (KD) of 2.6 × 10-8 M. In vitro analysis revealed that the administration of H2Mab-41 induced high levels of ADCC and CDC in CHO/dHER2 cells. Furthermore, intraperitoneal administration of H2Mab-41 in mouse xenograft models of CHO/dHER2 resulted in significant inhibition of tumor development compared to the control mouse IgG. Thus, the findings of the present study demonstrated the in vivo safety and efficacy of H2Mab-41, highlighting its suitability to be included as a part of a therapeutic regimen for dHER2-expressing canine cancers.