RABBIT MONOCLONAL ANTIBODIES ARE POWERFUL TOOLS FOR THE DETECTION OF BREAST CANCER -- ASSOCIATED MARKERS.

RABBIT MONOCLONAL ANTIBODIES ARE POWERFUL TOOLS FOR THE DETECTION OF BREAST CANCER ASSOCIATED MARKERS. Maria Frolkis, Hui Li, Kyra Cowan,Yaohuang Ke, Jonathan Villalobos, Weimin Zhu, Y. Peter Li. Maria Frolkis, Hui Li, Kyra Cowan,Yaohuang Ke, Jonathan Villalobos, Weimin Zhu, Y. Peter Li. Epitomics Inc., Burlingame, California .. www.epitomics.com Epitomics Inc., Burlingame, California www.epitomics.com
ABSTRACT
Rabbit monoclonal antibodies (RabMAbs) have recently been shown to play an important role as research and diagnostic reagents. One area where RabMAbs have demonstrated their superior performance is in immunohistochemical analysis of formalin fixed paraffin-embedded tissues based on their high affinity and sensitivity. We have successfully developed a large number of high quality rabbit monoclonal antibodies against difficult targets such as protein phosphorylation sites. In this paper, we present results of our rabbit monoclonal antibodies detecting Breast Cancer- associated antigens which are known to have a direct effect on both the treatment and behavior of breast cancer. RabMAbs against HER-2, p53, ER-alpha and AKT1 were evaluated in a variety of different types of paraffin-embedded breast cancer tissues. Affinity of our anti-HER-2 RabMAb was found to be 10 - 100 times higher than two commercially available Mouse Monoclonal and Rabbit Polyclonal antibodies. The detection level of HER-2 expression in paraffin-embedded human breast carcinoma tissues for RabMAb varied from 3 ng/ml to 0.5 ng/ml depending on the type of tumor. The intensity of HER-2 expression showed positive correlation with AKT1 and p53 immunostaining in tested breast cancer samples. We found that >90% of Grade III breast cancer tissues exhibited strong staining for phospho-AKT. Using anti-Phospho-Serine-118-E.R.-alpha RabMAb, it was determined that estrogen receptor alpha, specifically phosphorylated at Serine 118, is detectable in multiple human breast cancer tissues. Statistical analysis showed a negative correlation between HER-2 and ER-alpha expression in the same tumor tissues. Majority of breast cancer tissues were screened with Phospho anti-beta-Catenin RabMAb (Wnt signaling pathway) and RabMabs against main effectors of BCL cell signaling pathways. Immunohistochemical analysis of various paraffin-embedded tissues strongly correlated with cells immunofluorescent staining and Western Blot data. Here we demonstrate that Rabbit Monoclonal Antibody technology has the potential to create high affinity antibodies against key antigens in the pathogenesis of breast cancer.

Figure 1. Rabbit Monoclonal Antibodies Against Breast Cancer Markers

Figure 4. Expression of breast-cancer markers in different stages of Breast Cancer (Invasive Ductal Carcinoma)
HER-2 A. p53 ER-alpha PR

Figure 6. Phospho-specific RabMAbs: Anti-ER-alpha (pS118) RabMAb

Control LP block

A.

BCL-2
A B C D E

BAX

AKT

Rabbit Monoclonal Antibodies have the potential to create high affinity antibodies against key antigens in the pathogenesis of breast cancer: Immunohistochemical analysis of paraffin-embedded human breast carcinomas using : A- anti-HER-2 RabMAb, B- anti-ER-alpha RabMAb, C-anti-PR, D- anti-p53 RabMAb, E- anti-AKT RabMAb

B.
HER-2 B. p53 ER-alpha PR

Figure 2. Anti-HER-2 RabMAb

kDa 2501501007550372520-

BCL-2

BAX

AKT

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C. D.
90 80 70 60 50 40 30 20 10 0
%

kD
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1 Control

% of HER-2 expressing tumors

AKTexpression

100ER-alpha p53 AKT

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INTRODUCTION
Rabbit monoclonal antibodies (RabMAb) combine the advantage of high affinity, attributable to their rabbit origin, and high specificity due their monoclonal nature. The availability of the fusion partner, created in 1995 by Dr Katherine Knight (Loyola University, Chicago), enabled generation of stable hybridomas (Spieker-Polet et al., 1995. PNAS 92, 9348-52). The following year, Dr Robert Pytela and his colleagues established a robust system for the development of rabbit monoclonal antibodies. Using this proprietary RabMAb technology, we have successfully produced a large number of antibodies which perform well in immunohistochemical analysis of formalin fixed paraffin-embedded tissues. In this study, we present RabMAbs which detect Breast Cancer Associated antigens: HER-2, ER-alpha, PR, p53, AKT, BCL-2, BAX and AKT in breast cancer tissues We evaluated 34 samples of primary breast tumors. Tissues were rapidly collected, formalin-fixed and paraffin-embedded. The histology of every sample was uniformly interpreted by a pathologist in H&E-stained sections. Fluorescent immunocytochemistry was performed on chamber slides with routinely grown MCF-7 and SKBR3 cell lines. Extact aliquots of the same cells were analyzed by Western Blotting. Each tissue was analyzed at least 3 times with the same antibody at various antibody dilutions. We demonstrated that expression of the analyzed markers varies depending on tumor type and histological stage (TNM classification). Our results indicated for example that the intensity of HER-2 expression correlated with AKT1 and p53 immunostaining; 98% of Grade III breast cancer tissue exhibited strong staining for AKT1, however only 25% of Grade I tumor tissues showed AKT1 staining (Figure 4). An IHC comparison of several anti-HER-2 antibodies on formalin fixed tissues demonstrated that Epitomics anti-HER2 RabMAb could detect HER2 in much lower concentrations than currently commercially available Rabbit Polyclonal and Mouse Monoclonal antibodies (Figure 3). Phospho-specific antibodies have emerged as key tools for studying phosphorylation events by a variety of techniques including immunohistochemical analysis. L Murphy et al. (Clinical Cancer Research, 2004) showed the presence of ER-alpha specifically phosphorylated at SER 118 in human breast cancer biopsy samples. We demonstrated that Phospho-Specific anti-ER-alpha (pS118) RabMAb detects the phosphorylation site of ER-alpha in various breast tumor tissues by IHC (Figure 6). Immunohistochemical double staining with ER-alpha and HER-2 was conducted on all tissue samples of invasive breast cancer and ductal carcinoma in situ. HER-2 and ER-alpha expressions were inversely correlated in 65% of tested breast cancer tissues (Figure 5). In general, immunohistochemical analysis strongly correlated with immunofluorescent staining of cells and Western Blot data.

A. Immunofluorescent histochemistry of Ductal Carcinoma In Situ and B. Immunofluorescent staining of SKR3 cells using anti-HER-2 RabMAb C. Western blot analysis on Her2 transfected cell lysate using anti-ErbB2/HER2 RabMAb, dilution 1:1,000,000.

50Histological grade 3 Histological grade 1

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Non-phospho Peptide block

Figure 3. Anti-HER-2 RabMAb : Comparison with existing commercial antibodies

RabMAb Rab Polyclonal Mouse MAb

A. Example of expression of breast cancer - associated markers in Invasive ductal carcinoma, histological stage I B. Example of expression of breast cancer - associated markers in Invasive ductal carcinoma, histological stage III C. 40% of HER-2 expressing tumors express ER-alpha; 70% of HER-2 expressing tumors express p53 80% of HER-2 expressing tumors express AKT D. 98% of Histological grade 3 tumors express AKT compared with only 25 % of Histological grade 1 tumors.

252015-

Phospho Peptide block

Detection tool - Rabbit Monoclonal Antibodies

A. Immunofluorescent staining of untreated) and Phosphatase- treated MCF-7 cells using Phospho-ER-alpha RabMab B. Immunohistochemical staining of untreated and Phosphatase- treated paraffin-embedded Human Breast Adenocarcinoma using Phospho-ER-alpha RabMab C. Western Blott analysis of MCF7cells lysates using anti-Phospo-ER alpha antibody. 1. Control cell lysate., 2. Cell lysate treated with betta-Estradiol and EGF. D. Immunohistochemical analysis of paraffin-embedded Invasive Breast Carcinoma by antiPhospho-ER alpha RabMAb:: 1.- Control slide. 2.- Staining has not been blocked by incubation of RabMAb with Non-phospho-peptide. 3.- Staining was blocked by incubation of RabMAb with Phospho-peptide.

Figure 5. Dual IHC staining of Breast Cancer tissues using Anti-Mouse and Anti-Rabbit monoclonal antibodies
A.
B. C.

CONCLUSIONS
C

A. HER-2 expression in Ductal Carcinoma In Situ , concentration of all anti-HER-2 antibodies- 3 ng/ml B. HER-2 expression in Invasive Ductal Carcinoma stage III , concentration of all anti-HER-2 antibodies- 20 ng/ml C. HER-2 expression in Invasive Ductal Carcinoma stage II , concentration of all anti-HER-2 antibodies- 30 ng/ml

I. Nuclear (brown staining) of ER-alpha detected by anti-ER-alpha Mouse monoclonal antibody and anti-Mouse HRP conjugated secondary antibody. II. Membrane (pink staining) of HER-2 detected by anti-HER-2 Rabbit monoclonal antibody and anti-rabbit AP-conjugated secondary antibody.
A. Invasive Ductal Carcinoma expressing both ER-alpha and HER-2 B. Ductal Carcinoma In Situ expressing HER-2 only C. Invasive Ductal Carcinoma expressing ER-alpha only

1. RabMAbs against key antigens of breast cancer exhibited superior staining performance on formalin fixed paraffin-embedded breast tumor tissues. 2. Using RabMAbs, we detected correlation of HER-2, p53, AKT1 and ER-alpha expression among different types of breast cancer tissues. 3. We developed an anti-ER-alpha RabMAb against pS118 phosphorylation site which detects ER-alpha phosphorylation in various breast cancer tissues by IHC. 4. RabMAbs enable the possibility of clean dual color staining of tissue samples when paired with a quality Mouse moncolonal Ab.

Epitomics Inc., 863 Mitten Rd. suite 103, Burlingame, CA 94010-1303. Phone (650) 583-6688x223, FAX (650) 583-6680. peter.li@epitomics.com Epitomics Inc., 863 Mitten Rd. suite 103, Burlingame, CA 94010-1303. Phone (650) 583-6688x223, FAX (650) 583-6680. peter.li@epitomics.com