Global fitted (dotted line) SPR data of murine MX35 and Rebmab200 binding to immobilized synthetic NaPi2b epitope.

<p>MX35 (A) and Rebmab200 (B) were injected at concentrations ranging from 5 to 80 nM. After a 10 min association phase, the dissociation phase was followed for additional 10 min. Following double subtractive referencing, the curves were plotted using a 1∶1 Langmuir binding model, using Biacore T100 Evaluation Software. The solid line represents the experimental data and the dotted line the mathematical model for the binding of MX35 and Rebmab200 to the synthetic NaPi2b epitope.</p

Comparison of ADCC activity between MX35 and Rebmab200 (stable pool) over a range of mAb concentrations.

<p>The % of cytotoxicity represents antibody-mediated cell lysis measured by release of ⁵¹Cr from labeled ovarian cancer cells (OVCAR-3). Effector cells were obtained from donated human peripheral blood. Rebmab100 was used as a positive control, and Zenapax (Roche) was used as a negative control. The assay was repeated with MCF-7 cells, a NaPi2b negative and Le^Y positive (Rebmab100 antigen) tumor cell line, showing no ADCC results (data not shown).</p

Immunoglobulin HC and LC mRNA levels and productivity of the most representative Rebmab200 clones as determined by RT-qPCR.

<p><b>GAPDH was used as a reference gene.</b> Vector represents PER.C6®cells transfected with mock vector; PER.C6® corresponds to the parental cell. Error bars represent the standard deviation of triplicates. Qp represents specific productivity levels.</p

Immunoreactivity of MX35 and Rebmab200 in live cells by flow cytometry.

<p>Immunoreactivity of MX35 and Rebmab200 in live cells by flow cytometry.</p

Immunoreactivity of MX35 and Rebmab200 in tumor tissues by immunohistochemistry.

<p>Immunoreactivity of MX35 and Rebmab200 in tumor tissues by immunohistochemistry.</p

Comparison of the immuno-affinity of MX35 (left) at 1/50 dilution and Rebmab200 (right) at 1/100 dilution in parallel reactions using the same buffers and amplification and development conditions.

<p>Serial sections of serous papillary ovary adenocarcinoma (A), clear cell renal carcinoma (B), large cell poorly differentiated carcinoma of the lung (C) and invasive ductal carcinoma of the breast (D). Original magnification = 100×.</p

Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

<div><p>The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with ²¹¹At-Rebmab200. Here, the biodistribution of ²¹¹At-Rebmab200 was evaluated, as was the utility of ^99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its <i>in-vitro</i> capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and <i>in-vivo</i> tumor binding. We also demonstrated that ^99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.</p></div

Tissue distributions of ²¹¹At-MX35 and ²¹¹At-Rebmab200.

<p>^<b>211</b>At-MX35 (filled bars) and ^<b>211</b>At-Rebmab200 (open bars). Results are given as mean ± standard deviation of percent injected activity per gram tissue (%-IA/g). *P<0.05 by Student’s <i>t</i>-test. Abbreviation RBM L&R is the red bone marrow taken from left and right femur.</p

Representative transverse, coronal, and sagittal views from SPECT, micro-CT, and fused SPECT/CT.

<p>Tumor-bearing mice were imaged 24 h after injection of (A) 74 MBq of Rebmab200-HYNIC-^<b>99m</b>Tc or (B) control IgG1 antibody. Arrows indicate tumor masses.</p

Tissue distribution ratios^* of ¹²⁵I-MX35 and ¹²⁵I-RebmAb200 in nude mice.

<p>*Results are given as mean ± standard deviation of </p><p></p><p></p><p></p><p><mo stretchy="true">[</mo></p><p></p><p></p><p></p><p></p><p><mi>%</mi>IA</p><mo>/</mo><p>g<mo stretchy="false">(</mo></p><p>tissue</p><mo>)</mo><p></p><p></p><p></p><p></p><p></p><p><mi>%</mi>IA</p><mo>/</mo><p>g<mo stretchy="false">(</mo></p><p>blood</p><mo stretchy="false">)</mo><p></p><p></p><p></p><p></p><p></p><mo stretchy="true">]</mo><p></p><p><mi>I</mi><mo>−</mo><mi>R</mi><mi>e</mi><mi>b</mi><mi>m</mi><mi>A</mi><mi>b</mi><mn>200</mn></p><mrow></mrow><p><mn>125</mn></p><p></p><p></p><p><mo stretchy="true">[</mo></p><p></p><p></p><p></p><p></p><p><mi>%</mi>IA</p><mo>/</mo><p>g<mo stretchy="false">(</mo></p><p>tissue</p><mo stretchy="false">)</mo><p></p><p></p><p></p><p></p><p></p><p><mi>%</mi>IA</p><mo>/</mo><p>g<mo stretchy="false">(</mo></p><p>blood</p><mo stretchy="false">)</mo><p></p><p></p><p></p><p></p><p></p><mo stretchy="true">]</mo><p></p><p><mi>I</mi><mo>−</mo><mi>M</mi><mi>X</mi><mn>35</mn></p><mrow></mrow><p><mn>125</mn></p><p></p><p></p><p></p><p></p><p></p> for three animals at each point in time.<p></p><p>Tissue distribution ratios<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126298#t001fn001" target="_blank">^*</a> of ¹²⁵I-MX35 and ¹²⁵I-RebmAb200 in nude mice.</p