Monoclonal Antibodies in Immunodiagnosis and Immunotherapy最新文献

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C₉Mab-24 (rat immunoglobulin [IgG]_2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (K_D) of C₉Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10^-9 M and 4.7 × 10^-10 M, respectively. Results indicated that C₉Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG₁, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.

Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an important therapeutic target in various cancer types, including breast and gastric cancers. We previously developed an anti-HER2 monoclonal antibody (mAb), H₂Mab-77 (mouse IgG₁, kappa), which detects HER2 and dog HER2 (dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors and could be valuable as part of an antibody treatment regimen for them.

Monoclonal antibodies targeted against SARS-COV-2 have been recruited in the challenging treatment of COVID-19 with few clinically significant side effects. Casivirimab/imdevimab, a combination of monoclonal antibodies targeted against SARS-COV-2 spike protein, was shown to effectively prevent recently infected high-risk COVID-19 patients from developing severe disease. Its efficacy waned with the emergence of the resistant omicron variant in late 2021. We recorded the real-world effect of casivirimab/imdevimab on 116 high-risk COVID-19 patients. Cumulative need for hospitalization, mortality, new-onset disease, and reinfections was monitored. Casivirimab/imdevimab effect was independent from previous immunization. The cohort was further divided into two subgroups: patients infected during a delta variant prevalent period were more likely to become admitted but as likely to die than patients infected with the omicron variant, in support of its protective effect from clinical studies. Cumulative reinfection incidence in treated patients, interestingly, was lower than in the general population.

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C₂Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C₂Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C₂Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C₂Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.

CD10 is a cell surface metalloendopeptidase that cleaves and degrades many secreted physiologically active peptides by its enzymatic activity. Although CD10 expression has been found in various types of cells, its expression is increased in several cancers, including renal cancer. In this study, the antitumor activity of a novel anti-human CD10 monoclonal antibody (mAb) was investigated. A defucosylated mouse IgG_2a version of C₁₀Mab-31 (31-mG_2a-f) was created from an anti-CD10 mAb, C₁₀Mab-31 (IgG₁, kappa). Both C₁₀Mab-31 and 31-mG_2a-f specifically reacted with endogenous CD10 in renal cancer cells, VMRC-RCW, with the dissociation constant (K_D) values of 6.3 × 10^-9 M and 1.1 × 10^-9 M, respectively, indicating high binding affinity. To further examine the anti-CD10 mAb-mediated effector functions, the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were examined. The 31-mG_2a-f significantly exhibited ADCC and CDC against VMRC-RCW cells in vitro. Furthermore, 31-mG_2a-f exhibited antitumor activities in mouse xenografts of VMRC-RCW cells. These results suggest that 31-mG_2a-f exerts antitumor activities against CD10-expressing renal cancers and could be a valuable therapeutic candidate for treating them.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C₆Mab-13 (rat IgG₁, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (K_D) of C₆Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10^-9 M, indicating that C₆Mab-13 binds to mCCR6 with high affinity. In summary, C₆Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.