Potentiation of radiation-induced growth inhibition by letrozole measured by cell-count assay

<p><b>Copyright information:</b></p><p>Taken from "Letrozole sensitizes breast cancer cells to ionizing radiation"</p><p>Breast Cancer Research 2004;7(1):R156-R163.</p><p>Published online 7 Dec 2004</p><p>PMCID:PMC1064115.</p><p>Copyright © 2004 Azria et al., licensee BioMed Central Ltd.</p> Growth of MCF-7CA cells, measured for 18 days after treatment, was inhibited to a 76% greater extent with letrozole plus 4 Gy radiation after 12 days, and to an 85% greater extent after 18 days, compared with radiation alone. Solid lines, ■ and ◆ represent radiation alone at 2 Gy and 4 Gy, respectively; dotted lines, ■ and ◆ represent combination of radiation plus letrozole (0.7 μM) at 2 Gy and 4 Gy, respectively

Potentiation of radiation-induced growth inhibition by letrozole measured by the MTT assay

<p><b>Copyright information:</b></p><p>Taken from "Letrozole sensitizes breast cancer cells to ionizing radiation"</p><p>Breast Cancer Research 2004;7(1):R156-R163.</p><p>Published online 7 Dec 2004</p><p>PMCID:PMC1064115.</p><p>Copyright © 2004 Azria et al., licensee BioMed Central Ltd.</p> Growth of MCF-7CA cells, measured 6 days after treatment, was inhibited to a 40% greater extent with letrozole plus 2 Gy radiation, and to a 76% greater extent with letrozole plus 4 Gy radiation, compared with radiation alone

Potentiation of radiation-induced growth inhibition by letrozole measured by clonogenic assay

<p><b>Copyright information:</b></p><p>Taken from "Letrozole sensitizes breast cancer cells to ionizing radiation"</p><p>Breast Cancer Research 2004;7(1):R156-R163.</p><p>Published online 7 Dec 2004</p><p>PMCID:PMC1064115.</p><p>Copyright © 2004 Azria et al., licensee BioMed Central Ltd.</p> With radiation alone the MCF-7CA cell survival fraction decreased in a dose-dependent manner, which was significantly potentiated by the addition of 0.7 μM letrozole. For 2 Gy radiation, the surviving fraction was 0.66 with radiation alone and was 0.46 with the addition of letrozole (= 0.02). For 3 Gy radiation, the corresponding surviving fractions were 0.4 and 0.18, respectively (= 0.02)

Quantification of HER Expression and Dimerization in Patients’ Tumor Samples Using Time-Resolved Förster Resonance Energy Transfer

<div><p>Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with <em>EGFR</em> mRNA copy number (<em>P</em><0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients’ treatment in oncology.</p> </div

Comparison of our TR-FRET assay with standard techniques for the assessment of HER2 status.

<p>The number of HER2/cell was evaluated by TR-FRET in breast tumors stratified according to their Herceptest™ score and the <i>HER2</i> gene amplification status. <i>HER2</i> amplification was determined by FISH and confirmed by qPCR. The Herceptest™ scoring system was as follows: 0, no staining or membrane staining in <10% of tumor cells; 1+, faint/barely perceptible membrane staining in >10% of tumor cells; 2+: weak to moderate complete membrane staining in >10% of tumor cells, or strong complete membrane staining in 10–30% of tumor cells; 3+, strong complete membrane staining in >30% of tumor cells.</p

Characterization of the tumor xenograft models using the developed antibody-based TR-FRET assays.

<p>(a) Absolute quantification of EGFR and HER2 expression in NIH/3T3 EGFR (high EGFR expression, no HER2 expression), NIH/3T3 HER2 (high HER2 expression, no EGFR expression), NIH/3T3 EGFR/HER2 (high EGFR and HER2 expression) and SKOV-3 (low EGFR expression, high HER2 expression) tumor xenografts using the HER quantification assays. (b) Detection of EGFR:EGFR, HER2:HER2 and EGFR:HER2 dimers using the HER dimer quantification assays.</p

Quantification of HER dimers using the TR-FRET technology.

<p>When an antibody conjugated with Lumi4® Tb (donor fluorophore) and an antibody coupled to d2 (acceptor fluorophore) are brought together by a biological interaction, a portion of the energy captured by Lumi4® Tb during excitation is released through fluorescence emission, while the remaining energy is transferred to d2, which re-emits a specific long-lived fluorescence at 665 nm. In the case of homodimer quantification, the fluorescence emitted will represent only 50% of the actual amount of homodimers due to the formation also of homodimers that interact with two antibodies that are both labeled with the donor or the acceptor fluorophore and that are, therefore, undetectable.</p

Correlation between TR-FRET and RT-qPCR measurements of EGFR expression.

<p>EGFR protein expression determined by TR-FRET was plotted against <i>EGFR</i> mRNA copy number measured by RT-qPCR. With a determination coefficient (R²) of 0.73, the resulting curve demonstrates a linear correlation between the measurements of the two assays.</p

Table_1_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p

Table_4_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p