{"title":"Experimental Determination of Antibody Affinity and Avidity: Guidance and Considerations.","authors":"Andrei Moroz, Cory L Brooks","doi":"10.1089/mab.2024.0019","DOIUrl":"https://doi.org/10.1089/mab.2024.0019","url":null,"abstract":"","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142395233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Coagulation factor XIII (FXIII) is an enzyme that strengthens hemostatic clots, and its deficiency can cause life-threatening bleeding. We immunized mice with human plasma-derived FXIII to generate monoclonal antibodies (mAbs) against the B subunit (FXIII-B), which stabilizes the A subunit (FXIII-A) of FXIII, and analyzed their properties. The epitopes of the seven mouse antihuman FXIII-B mAbs obtained were found to be the 3rd, 5th, 6th, 9th, and 10th Sushi domains. One of these mAbs, mAb 5-6C, recognized the 10th Sushi domain and inhibited the fibrin cross-linking reaction without affecting the amine incorporation activity of FXIII. We previously reported that the 10th Sushi domain is the site where FXIII-B binds to fibrin and functions to bring FXIII-A closer to the substrate fibrin. Except for mAb 5-6C, mouse mAbs with high yields were used to measure the amount of FXIII-B antigen by an immunochromatography test (ICT), which showed a high correlation with enzyme-linked immunosorbent assay-obtained results. In addition, we developed a prototype ICT to detect anti-FXIII-B autoantibodies using mAb 1-3C, which showed good results in measuring the amount of FXIII-B antigen. Thus, mouse mAbs may be useful for clinical applications. mAb 5-6C targeting the 10th Sushi domain may also be useful for inhibiting thrombosis progression when humanized as antibody medicines.
凝血因子 XIII(FXIII)是一种强化止血凝块的酶,缺乏这种酶可导致危及生命的出血。我们用人血浆提取的 FXIII 对小鼠进行免疫,产生了针对 B 亚基(FXIII-B)的单克隆抗体(mAbs),该抗体能稳定 FXIII 的 A 亚基(FXIII-A),并分析了它们的特性。研究发现,获得的七种小鼠抗人 FXIII-B mAbs 的表位分别是第 3、5、6、9 和 10 Sushi 结构域。其中一种 mAb 5-6C 能识别第 10 个 Sushi 结构域,并抑制纤维蛋白交联反应,但不影响 FXIII 的胺结合活性。我们以前曾报道,第 10 个 Sushi 结构域是 FXIII-B 与纤维蛋白结合的部位,其功能是使 FXIII-A 靠近底物纤维蛋白。除 mAb 5-6C 外,我们还利用产量较高的小鼠 mAb 通过免疫层析检测(ICT)来测量 FXIII-B 抗原的含量,其结果与酶联免疫吸附试验的结果高度相关。此外,我们还开发了一种利用 mAb 1-3C 检测抗 FXIII-B 自身抗体的原型 ICT,在测量 FXIII-B 抗原量方面显示出良好的效果。以第 10 个 Sushi 结构域为靶点的 mAb 5-6C 在人源化成为抗体药物后,也可用于抑制血栓形成。
{"title":"Development and Epitope Mapping of Seven Mouse Anti-Human Coagulation Factor XIII-B Subunit Monoclonal Antibodies.","authors":"Tsukasa Osaki, Yasuo Magari, Masayoshi Souri, Akitada Ichinose","doi":"10.1089/mab.2024.0016","DOIUrl":"https://doi.org/10.1089/mab.2024.0016","url":null,"abstract":"<p><p>Coagulation factor XIII (FXIII) is an enzyme that strengthens hemostatic clots, and its deficiency can cause life-threatening bleeding. We immunized mice with human plasma-derived FXIII to generate monoclonal antibodies (mAbs) against the B subunit (FXIII-B), which stabilizes the A subunit (FXIII-A) of FXIII, and analyzed their properties. The epitopes of the seven mouse antihuman FXIII-B mAbs obtained were found to be the 3rd, 5th, 6th, 9th, and 10th Sushi domains. One of these mAbs, mAb 5-6C, recognized the 10th Sushi domain and inhibited the fibrin cross-linking reaction without affecting the amine incorporation activity of FXIII. We previously reported that the 10th Sushi domain is the site where FXIII-B binds to fibrin and functions to bring FXIII-A closer to the substrate fibrin. Except for mAb 5-6C, mouse mAbs with high yields were used to measure the amount of FXIII-B antigen by an immunochromatography test (ICT), which showed a high correlation with enzyme-linked immunosorbent assay-obtained results. In addition, we developed a prototype ICT to detect anti-FXIII-B autoantibodies using mAb 1-3C, which showed good results in measuring the amount of FXIII-B antigen. Thus, mouse mAbs may be useful for clinical applications. mAb 5-6C targeting the 10th Sushi domain may also be useful for inhibiting thrombosis progression when humanized as antibody medicines.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This Long-Covid disease, mild or severe, is multiorgan or system-wide, spanning from fatigue to clotting abnormalities and autoantibody. The spectrum of different symptoms in Long-Covid diseases makes it difficult to point to a common immunopathogenic etiology. Different immune pathways are presented and critically evaluated. A hypothesis is advanced that indicates autoimmune reactions as cause for Long-Covid disease. The immune network pathway describes a redirection of the nominal anti-SARS-CoV response towards an autoimmune target. Several therapeutic interventions are suggested to suppress the autoimmune pathway.
{"title":"Immune Jumping in Autoimmune Long-Covid.","authors":"Heinz Kohler","doi":"10.1089/mab.2024.0006","DOIUrl":"https://doi.org/10.1089/mab.2024.0006","url":null,"abstract":"<p><p>This Long-Covid disease, mild or severe, is multiorgan or system-wide, spanning from fatigue to clotting abnormalities and autoantibody. The spectrum of different symptoms in Long-Covid diseases makes it difficult to point to a common immunopathogenic etiology. Different immune pathways are presented and critically evaluated. A hypothesis is advanced that indicates autoimmune reactions as cause for Long-Covid disease. The immune network pathway describes a redirection of the nominal anti-SARS-CoV response towards an autoimmune target. Several therapeutic interventions are suggested to suppress the autoimmune pathway.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142156599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.
磷酸核糖基氨基咪唑羧化酶、磷酸核糖基氨基咪唑琥珀酰甲酰胺合成酶(PAICS)是一种新的嘌呤生物合成酶。已发现它在各种癌症中过度表达,并与细胞增殖、侵袭、上皮-间质转化和肿瘤的高效生长有关。在本研究中,我们描述了一种大鼠单克隆抗体(mAb)6A10,它是作为人类 PAICS 的抗原生成的。这种 mAb 的产生是为了与人 PAICS 的 N 端区域相互作用,并且发现它能识别几种癌细胞中的内源性 PAICS 酶。我们的结果还表明,它能识别与人类 PAICS 抗原区氨基酸序列相同的猴和狗 PAICS,但不能识别大鼠和小鼠 PAICS。此外,我们的数据还表明,这种 mAb 适合用于几种癌细胞系的免疫沉淀和免疫印迹。因此,我们预计 mAb 6A10 将有助于人类 PAICS 在多种癌症中的功能分析以及恶性转化的诊断。
{"title":"Generation of a Rat Monoclonal Antibody for Human PAICS, a <i>de novo</i> Purine Biosynthetic Enzyme.","authors":"Chikako Yokoyama, Kaori Bando, Momoka Yamagata, Takeshi Nagasaki, Taro Tachibana","doi":"10.1089/mab.2023.0030","DOIUrl":"10.1089/mab.2023.0030","url":null,"abstract":"<p><p>Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a <i>de novo</i> purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the <i>N</i>-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141248954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01Epub Date: 2024-07-22DOI: 10.1089/mab.2024.0008
Bailin Tu, Zhihong Lin, Jeff Moore, Archana Krishnan Sekaran, Miranda J Wesley, De Yu Mao, Mark Gibson, Wan-Ching Lai, John Boggs, Thomas Slowik, Yeni Natalia C Perez-Gelvez, Ryan Bonn, Tracey Rae, Jeremy Minshull, Ferenc Boldog, Varsha Sitaraman, Scott Muerhoff, Philip Hemken
Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.
{"title":"Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.","authors":"Bailin Tu, Zhihong Lin, Jeff Moore, Archana Krishnan Sekaran, Miranda J Wesley, De Yu Mao, Mark Gibson, Wan-Ching Lai, John Boggs, Thomas Slowik, Yeni Natalia C Perez-Gelvez, Ryan Bonn, Tracey Rae, Jeremy Minshull, Ferenc Boldog, Varsha Sitaraman, Scott Muerhoff, Philip Hemken","doi":"10.1089/mab.2024.0008","DOIUrl":"10.1089/mab.2024.0008","url":null,"abstract":"<p><p>Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and <i>in vitro</i> diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141735648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01Epub Date: 2024-06-05DOI: 10.1089/mab.2024.0002
Hiyori Kobayashi, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato
The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.
C-C motif趋化因子受体8(CCR8)在调节性T(Treg)细胞中高选择性表达,与肿瘤进展有关。Treg细胞在肿瘤中的大量聚集抑制了CD8+细胞对肿瘤细胞的效应功能。因此,使用抗CCR8单克隆抗体(mAbs)选择性地清除Treg细胞可重振抗肿瘤免疫反应,改善对癌症免疫疗法的反应。此前,我们利用细胞免疫和筛选方法开发了一种抗小鼠CCR8(mCCR8)mAb--C8Mab-2。本研究使用流式细胞术研究了 C8Mab-2 的结合表位。mCCR8 细胞外结构域替代突变体分析表明,C8Mab-2 能识别 mCCR8 的 N 端区域(1-33 个氨基酸)。接着,在 N 端区域进行了 1×丙氨酸(或甘氨酸)扫描和 2×丙氨酸(或甘氨酸)扫描。结果显示,17-DFFTAP-22 序列对 C8Mab-2 的识别非常重要,而 Thr20 是表位的中心氨基酸。这些结果揭示了 mCCR8 的 N 端参与了 C8Mab-2 的识别。
{"title":"Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C<sub>8</sub>Mab-2 Using Flow Cytometry.","authors":"Hiyori Kobayashi, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato","doi":"10.1089/mab.2024.0002","DOIUrl":"10.1089/mab.2024.0002","url":null,"abstract":"<p><p>The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8<sup>+</sup> cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C<sub>8</sub>Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C<sub>8</sub>Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C<sub>8</sub>Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the <sub>17-</sub>DFFTAP<sub>-22</sub> sequence is important for the recognition by C<sub>8</sub>Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C<sub>8</sub>Mab-2.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141248953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01Epub Date: 2024-06-05DOI: 10.1089/mab.2024.0004
Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Tsunenori Ouchida, Mika K Kaneko, Yukinari Kato
C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.
{"title":"Development of a Sensitive Anti-Mouse CCR5 Monoclonal Antibody for Flow Cytometry.","authors":"Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Tsunenori Ouchida, Mika K Kaneko, Yukinari Kato","doi":"10.1089/mab.2024.0004","DOIUrl":"10.1089/mab.2024.0004","url":null,"abstract":"<p><p>C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C<sub>5</sub>Mab-2 (rat IgG<sub>2b</sub>, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (<i>K</i><sub>D</sub>) of C<sub>5</sub>Mab-2 for CHO/mCCR5 was determined as 4.3 × 10<sup>-8</sup> M. These results indicated that C<sub>5</sub>Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141248947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01Epub Date: 2024-06-13DOI: 10.1089/mab.2024.0009
Rena Ubukata, Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Mika K Kaneko, Yukinari Kato
One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.
{"title":"Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization.","authors":"Rena Ubukata, Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Mika K Kaneko, Yukinari Kato","doi":"10.1089/mab.2024.0009","DOIUrl":"10.1089/mab.2024.0009","url":null,"abstract":"<p><p>One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C<sub>5</sub>Mab-4 (rat IgG<sub>2a</sub>, kappa) and C<sub>5</sub>Mab-8 (rat IgG<sub>1</sub>, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (<i>K</i><sub>D</sub>) values of C<sub>5</sub>Mab-4 and C<sub>5</sub>Mab-8 for CHO/mCCR5 were determined as 3.5 × 10<sup>-8</sup> M and 7.3 × 10<sup>-9</sup> M, respectively. Furthermore, both C<sub>5</sub>Mab-4 and C<sub>5</sub>Mab-8 could detect mCCR5 by western blotting. These results indicated that C<sub>5</sub>Mab-4 and C<sub>5</sub>Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141312337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-03-20DOI: 10.1089/mab.2023.0024
Tsunenori Ouchida, Yu Isoda, Tomohiro Tanaka, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato
C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.
{"title":"Cx<sub>3</sub>Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal Antibody for Flow Cytometry.","authors":"Tsunenori Ouchida, Yu Isoda, Tomohiro Tanaka, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato","doi":"10.1089/mab.2023.0024","DOIUrl":"10.1089/mab.2023.0024","url":null,"abstract":"<p><p>C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx<sub>3</sub>Mab-4 (rat IgG<sub>1</sub>, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx<sub>3</sub>Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx<sub>3</sub>Mab-4 was determined as 1.3 × 10<sup>-9</sup> M, indicating that Cx<sub>3</sub>Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx<sub>3</sub>Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.</p>","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140177676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-06-10DOI: 10.1089/mab.2024.0012
Cory L Brooks, Andrei Moroz
{"title":"Charting the Course for <i>Monoclonal Antibodies in Immunodiagnosis and Immunotherapy</i>.","authors":"Cory L Brooks, Andrei Moroz","doi":"10.1089/mab.2024.0012","DOIUrl":"10.1089/mab.2024.0012","url":null,"abstract":"","PeriodicalId":53514,"journal":{"name":"Monoclonal Antibodies in Immunodiagnosis and Immunotherapy","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}