Boyang Chu

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.

Abstract 415: A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC

All non-small cell lung cancer (NSCLC) patients are recommended to be screened for anaplastic lymphoma kinase (ALK)-rearrangement despite its low occurrence ( Citation Format: Hsiangmin Lu, Rachel Gonzalez, Yi Shen, Mu-lan Jin, Yipan Wu, Yungang Zhang, Kehu Yuan, Boyang Chu, Lili Qi, Huibo Liu, Chenlin Wang, Guangli Wang, Youmin Shu, Julie McDowell, Donghui Ma, Wei-wu He, Jian Chen, Ray Lin. A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 415.

Abstract 3393: Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology

Programmed death-1 (PD-1) expresses in many tumors in response to inflammation. It is up-regulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands PD-L1 and PDL2 and serves as a key checkpoint of the immune system. Pembrolizumab is the first anti-PD-1 therapy approved in the United States and received FDA9s Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin-fixed paraffin-embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed. To develop the best monoclonal antibody for PD-1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono-specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross-reactivity tests were also explored. In summary, two newly generated monoclonal antibodies demonstrated ultra-specificity against PD-1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before pembrolizumab prescription. Citation Format: Caiwei Chen, Yanlin Tang, Haitao Wei, Kehu Yuan, Guiyin Wu, Jian Chen, Boyang Chu, Guangli Wang, Youmin Shu, Wei-Wu He, Donghui Ma. Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2015-3393

Abstract 3386: The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry

Ki-67 is a nuclear protein that is a useful marker of proliferation. Ki-67 antibodies are used to generate a Ki-67 labeling index, which is the percentage of cells with nuclear immunostaining in immunohistochemistry. The Ki-67 labeling index may be useful as a prognostic and predictive marker in cancer. The Ki-67 labeling index relies on the use of antibodies with a suitable level of sensitivity and specificity, and many Ki-67 antibodies have been developed over the last two decades. One of the most widely used Ki-67 antibodies is the mouse monoclonal MIB1. However, there have been many reports showing that in some cases MIB1 detects an unexpected membranous and cytoplasmic staining pattern in immunohistochemistry. Since Ki-67 is exclusively expressed in the nucleus this raises questions about the specificity of MIB1. We have created a nearly comprehensive microarray of about 17,000 human proteins to investigate the specificity of MIB1 and identify cross reacting proteins. The protein microarray was also used to screen new panels of mouse monoclonals against Ki-67. We have identified the antibody UMAB107 that in immunohistochemistry performs with similar sensitivity as MIB1, but unlike MIB1, UMAB107 is highly specific. Citation Format: Caiwei Chen, Kehu Yuan, Boyang Chu, Youmin Shu, Jian Chen, Joe Stafford, Wei Fu, Wei-Wu He, Ross Chambers, Donghui Ma. The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3386. doi:10.1158/1538-7445.AM2015-3386

Abstract 3399: Using protein chips to develop a highly specific HER2 antibody for HER2 amplification testing

Human epidermal growth factor receptor 2 (HER2) is an orphan receptor tyrosine kinase member of the EGFR families and is found to be a key tumor driver gene. In breast cancer and gastric cancer, HER2 amplification can be effectively treated by its neutralizing antibody, trastuzumab. In clinic, the HER2 immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted to another family member HER4. IHC staining also indicated that it has strong non-specific cytoplasmic and nucleus staining in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have successfully identified and confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and unclear staining of 4B5 on normal gastric epithelium. Finally, we have developed a novel HER2 mouse monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. UMAB36 could provide an alternative high specific IHC reagent for HER2 amplification testing in gastric cancer population. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Qi, Zhongwu Li, Guiyin Wu, Xiaozheng Huang, Yi Shen, Min Zhao, Wei Fu, Huibo Liu, Boyang Chu, Guangli Wang, Youmin Shu, Donghui Ma, Wei-Wu He, Jian Chen. Using protein chips to develop a highly specific HER2 antibody for HER2 amplification testing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2015-3399