Local Delivery of Nimustine Hydrochloride against Brain Tumors: Basic Characterization Study.

Convection-enhanced delivery (CED) delivers agents directly into tumors and the surrounding parenchyma. Although a promising concept, clinical applications are often hampered by insufficient treatment efficacy. Toward developing an effective CED-based strategy for delivering drugs with proven clinical efficacy, we performed a basic characterization study to explore the locally delivered characteristics of the water soluble nitrosourea nimustine hydrochloride (ACNU). First, ACNU distribution after CED in rodent brain was studied using mass spectrometry imaging. Clearance of 14C-labeled ACNU after CED in striatum was also studied. ACNU was robustly distributed in rodent brain similar to the distribution of the hydrophilic dye Evans blue after CED, and locally delivered ACNU was observed for over 24 h at the delivery site. Subsequently, to investigate the potential of ACNU to induce an immunostimulative microenvironment, Fas and transforming growth factor-ß1 (TGF-ß1) was assessed in vitro. We found that ACNU significantly inhibited TGF-ß1 secretion and reduced Fas expression. Further, after CED of ACNU in 9L-derived intracranial tumors, the infiltration of CD4/CD8 lymphocytes in tumors was evaluated by immunofluorescence.CED of ACNU in xenografted intracranial tumors induced tumor infiltration of CD4/CD8 lymphocytes. ACNU has a robust distribution in rodent brain by CED, and delayed clearance of the drug was observed at the local infusion site. Further, local delivery of ACNU affects the tumor microenvironment and induces immune cell migration in tumor. These characteristics make ACNU a promising agent for CED.

Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6Mab-1) Using the 2 × Alanine Scanning Method.

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, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.