Monoclonal Antibodies in Immunodiagnosis and Immunotherapy最新文献

The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells, including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C₂Mab-9 (mouse IgG₁, kappa), using the synthetic peptide immunization method. Flow cytometric and immunocytochemical results showed that C₂Mab-9 reacted with hCCR2-expressing U937 (human histiocytic lymphoma) and natural killer cells. Furthermore, C₂Mab-9 showed the moderate binding affinity for both cells. Conclusively, C₂Mab-9 can be a useful tool for analyzing hCCR2-related biological responses.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells in tumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx₆Mab-1 (rat IgG₁, kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (K_D) of Cx₆Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 × 10^-9 M, 3.4 × 10^-7 M, and 3.8 × 10^-7 M, respectively. Furthermore, Cx₆Mab-1 could detect endogenous mCXCR6 in P388 and J774-1 cells by Western blotting. These results indicated that Cx₆Mab-1 is useful for detecting mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing cells in vivo experiments.

T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is one of the immune checkpoint molecules. TIGIT is expressed in T or natural killer (NK) cells and is upregulated in several cancers. Because TIGIT suppresses the antitumor activity of the T or NK cells by binding to its ligand, such as CD155, CD112, and CD113, TIGIT can be a molecular marker or a therapeutic target for cancer immunotherapy. We previously developed an anti-human TIGIT (hTIGIT) monoclonal antibody (mAb; clone TgMab-2; mouse IgG₁, kappa) by the Cell-Based Immunization and Screening method. TgMab-2 binds to hTIGIT with high binding affinity in flow cytometry. In this study, we investigated the availability of TgMab-2 and its recombinant mAb (recTgMab-2) in immunocytochemistry. We found that TgMab-2 and recTgMab-2 bind to hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells, but not parental CHO-K1 cells, indicating that both mAbs specifically recognize hTIGIT. Furthermore, both mAbs recognized endogenous hTIGIT expressed in human NK cells in immunocytochemistry. These results demonstrate that TgMab-2 and recTgMab-2 are applicable for immunocytochemistry against hTIGIT.

Therapeutic agents targeting B cells, such as rituximab, have been proven to be effective in several diseases. However, since this therapy targets the whole B cell population, there are still concerns about critical adverse events. Therefore, a new type of antibody that can specifically deplete a particular type of B cell may have the potential to become an efficient and safer therapy. Membrane-bound IgA1 (mIgA1) is expressed on soluble IgA1 (sIgA1) producing B cells and/or their precursor, B cells. Although most of the amino acid sequence in the N-terminal regions of the extracellular domains of mIgA1 and sIgA1 is shared, there is a membrane type-specific region, named the membrane-bound immunoglobulin isotype-specific (migis-α) region, at the membrane-proximal region of mIgA1. We hypothesized that the migis-α region would be a suitable antigen for therapeutic antibodies to target mIgA1-expressing B cells without binding to sIgA1, which may cause undesired adverse effects and poor pharmacokinetics (PK). We established two anti-migis-α monoclonal antibodies (mAbs), KM4641 and KM4644, by immunization of the synthetic peptide corresponding to an migis-α region. These mAbs both showed robust binding to mIgA1-expressing transfectant cells. As we expected, neither mAbs bound to sIgA1 and we found that the mAbs recognized different seven to eight amino acid sequences within the migis-α region. Furthermore, the rat-human chimeric type of both mAbs showed antibody-dependent cellular cytotoxicity against mIgA1-expressing transfectant cells. Taken together, this study showed that established mAbs have therapeutic potential in IgA-related diseases, such as IgA nephropathy.