A novel immunotoxin reveals a new role for CD321 in endothelial cells

<div><p>There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.</p></div

Redistribution of CD321 during hypoxia.

<p>Immunocytochemical analysis of CD321 localization. SVEC4-10 cells were incubated under normoxic (pO₂ = 20%) or hypoxic (pO₂ = 1%) conditions for three days. Cells were fixed and stained with DAPI (blue), 90G4 antibody (green), and anti-SM22α antibody (red). Images were taken at (A) low or (B) high magnification. Scale bar; 50 μm.</p

Targeted cytotoxicity of the 90G4 immunotoxin in migratory cells.

<p>Live imaging of 90G4 immunotoxin administrated either (A–C) concomitantly or (D–F) one day after SVEC4-10 cell seeding. For each condition, samples were seeded in triplicate. (B, E) Phase contrast images were taken every 3 h and analyzed to calculate confluency (%). Values and error bars correspond to mean and standard error, respectively. (C, F) Images were taken at the indicated time during live imaging.</p

90G4 monoclonal antibody recognizes the CD321 antigen.

<p>(A) Expression profile in endothelial cell lines. Indicated endothelial cell lines were tested by staining with control (gray) or 90G4 antibodies (black). Data of FITC fluorescent intensity (FL1) indicated in histogram. Data presents three independent experiments. (B) Biochemical profiling of the molecular weight of the putative antigen. SVEC4-10 cells were surface-biotinylated with Sulfo-NHS-Biotin and lysed in NP40 buffer, followed by immunoprecipitation with 90G4 or IgG2a, к control antibodies. After SDS-polyacrylamide gel electrophoresis (PAGE), the putative antigen was visualized by probing the streptavidin-HRP (Str-HRP) conjugate by chemiluminescence. The molecular weight of the detected protein was 35 kDa. (C) Identification of 90G4 antigen. Amino acid sequence of CD321 proteins were indicated with the two of underlined peptide sequences that are detected by LC-MS/MS analysis. (D) Specific immunoreactivity of 90G4 antibody against CD321. The expression vectors encoding CD321, CD322, or CD323 cDNA were transfected into CHO cells, which were then subjected to flow cytometry analysis. Data are presented in contour plot (top) or overlaid in histogram (bottom) of FITC signal intensity of sample (black) or control (gray).</p

90G4 monoclonal antibody displays immunotoxin activity.

<p>(A) Principle of the DT3C-mediated immunotoxin screening. The antibody forms an immunocomplex with DT3C via the Fc-binding domain. If the immunocomplex bound to the cell surface is internalized with the target antigen, cleavage of the DT3C catalytic domain by the intracellular furin protease results in cytotoxicity. DT3C with either purified 90G4 or isotype control (iso; IgG2a, к) antibodies was administered to (B) MS-1 or (C) SVEC4-10 cells. After three days, cell viability was measured with the WST-1 assay. Data represent the mean value of triplicate samples for three independent experiments. Error bars correspond to the standard error of the mean.</p

Novel Hybrid Compound of a Plinabulin Prodrug with an IgG Binding Peptide for Generating a Tumor Selective Noncovalent-Type Antibody–Drug Conjugate

Although several approaches for making antibody–drug conjugates (ADC) have been developed, it has yet to be reported that an antibody binding peptide such as Z33 from protein A is utilized as the pivotal unit to generate the noncovalent-type ADC (NC-ADC). Herein we aim to establish a novel probe for NC-ADC by synthesizing the Z33-conjugated antitumor agent, plinabulin. Due to the different solubility of two components, including hydrophobic plinabulin and hydrophilic Z33, an innovative method with a solid-supported disulfide coupling reagent is required for the synthesis of the target compounds with prominent efficiency (29% isolated yield). We demonstrate that the synthesized hybrid exhibits a binding affinity against the anti-HER2 antibody (Herceptin) and the anti-CD71 antibody (6E1) (<i>K</i>_d = 46.6 ± 0.5 nM and 4.5 ± 0.56 μM, respectively) in the surface plasmon resonance (SPR) assay. In the cell-based assays, the hybrid provides a significant cytotoxicity in the presence of Herceptin against HER2 overexpressing SKBR-3 cells, but not against HER2 low-expressing MCF-7 cells. Further, it is noteworthy that the hybrid in combination with Herceptin induces cytotoxicity against Herceptin-resistant SKBR-3 (SKBR-3HR) cells. Similar results are obtained with the 6E1 antibody, suggesting that the synthesized hybrid can be widely applicable for NC-ADC using the antibody of interest. In summary, a series of evidence presented here strongly indicate that NC-ADCs have high potential for the next generation of antitumor agents