Monoclonal Antibodies in Immunodiagnosis and Immunotherapy最新文献

By converting extracellular adenosine triphosphate to adenosine, CD39 is involved in adenosine metabolism. The extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment. Therefore, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is thought to be one of the important strategies for tumor therapy. In this study, we developed novel mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCD39 mAbs, C₃₉Mab-2 (rat IgG_2a, lambda), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) and an endogenously mCD39-expressed cell line (SN36) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (K_D) values of C₃₉Mab-2 for CHO/mCD39 and SN36 were 5.5 × 10^-9 M and 4.9 × 10^-9 M, respectively. These results indicated that C₃₉Mab-2 is useful for the detection of mCD39 in flow cytometry.

Graves' disease is the most common cause of thyrotoxicosis and is characterized by ophthalmopathy with proptosis, chemosis, or conjunctival injection; pretibial myxedema; and thyroid acropachy. It is an autoimmune disease that can be genetic or influenced by coexisting environmental factors such as exposure to anticancer drugs, including immune checkpoint inhibitors. The incidence rate of breast cancer is increasing due to rising awareness of risk factors and screening for breast cancer, and the mortality rate is decreasing due to recent advances in cancer treatment. However, there are side effects that are attributed to these treatment modalities, manifesting in various forms in breast cancer survivors, which are reflected in the patient in this case study.

Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated. Clinical Trial Registration: clinicaltrials.gov NCT05415137.

Nattokinase is a protease produced by Bacillus subtilis var. natto that exhibits various beneficial biological effects. Thus, a reliable assay to determine nattokinase levels is needed. In this study, we developed novel mouse monoclonal antibodies (mAbs) that recognize nattokinase, and created a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure nattokinase levels. The ELISA was developed using a combination of new mouse antinattokinase mAbs used as capture antibodies coated onto 96-well plates, with a peroxidase-conjugated antibody used for detection. This ELISA enabled detection of nattokinase at 1 ng/mL. We believe that the novel mAbs developed in this study will be useful in future for elucidating nattokinase function.

Porcine transmissible gastroenteritis virus (TGEV) infection results in severe gastrointestinal disease manifesting vomiting, diarrhea in neonatal porcine, with extremely high mortality. Monoclonal antibody (MAb) specific to TGEV nonstructural protein (NSP)14 that contains two functional domains, exonuclease (ExoN) and methyltransferase (MTase) domains, may help elucidate the role of NSP14 in the viral life-cycle. In this study, we developed a murine MAb, designated 12F1, against TGEV NSP14 using traditional cell-fusion technique. It was shown the MAb can exclusively bind to viral NSP14, as evidenced by the results of indirect fluorescent assay and western blotting. Intriguingly, epitope screening assay shown that 12F1 targets a hinge region connecting ExoN and N7-MTase of NSP14.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinase family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, specific monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop specific anti-EphB4 mAbs for multiple applications using the Cell-Based Immunization and Screening method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, Western blot, and immunohistochemistry (IHC). B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell line, MCF-7, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (K_D) values were determined to be 2.9 × 10^-9 M and 1.3 × 10^-9 M by flow cytometric analysis for CHO/EphB4 and MCF-7 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in Western blot, and stained breast cancer tissues in IHC. Altogether, B4Mab-7 is very useful for detecting EphB4 in various applications.