Guangli Wang

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 1563: Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers

Published studies show tumor cells overexpress PD-L1 to escape the host immune defense by binding to PD-1 on surveilling T-cells causing the T-cells to shut down. Immunotherapies disrupting PD-1/PD-L1 signaling interactions have great success in treating PD-L1 positive tumors. Immunohistochemistry (IHC) is an important diagnostic tool currently used to determine the expression level of PD-L1 in tumor and immune cells. The FDA approved PD-L1 clones (SP142 and 28-8) for IHC present different staining patterns when evaluated on the same tumor tissue. In this study, we assessed both the immune cells and tumor cells IHC positive stain of PD-L1. To do this we evaluated multiple immune cell markers with multiple PD-L1 antibodies. IHC screens were done with 5 PD-L1 antibodies, 2 recombinant rabbit monoclonal antibodies (clone OR-5E3 and OR-5H8), the mouse monoclonal antibody (clone UMAB229), and the FDA approved clones (SP142 and 28-8). The immune cell markers used were CD3, CD8A, CD20, CD68, and FOXP3. NSCLC, Bladder Cancer, and Melanoma immune cells, as indicated by the CD3, CD8A, CD20, CD68 and FOXP3 staining, generated different distribution pattern in the three tumor types. The five PD-L1 antibodies did show variation in detection of both the immune and tumor cells. For example, PD-L1 clones OR-5E3 and OR-5H8 stained tumor cells stronger and picked up weak expression of PD-L1 better than clone SP142, suggesting the two antibodies have higher affinity. The mouse monoclonal anti-PDL1 clone UMAB229 picked up strong and weak staining similar or better than clones OR-5H8 and picked up the immune cells similar to SP-142. The five PD-L1 antibodies were made from different antigens which may contribute to their sensitivity and specificity to detect PD-L1 in tumor cells. This study suggests the new generation of PD-L1 antibodies may make a better tool for diagnostic screens. Citation Format: Rachel M. Gonzalez, Wei Fu, Evelin Logis, Emma Ding, Casey Chen, Xiaojie Li, Sonia Merritt, Mingjuan Liu, Amy Zhang, Yiran Wang, Guangli Wang, Donghui Ma. Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1563.

Abstract 3921: A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein

Increased sensitivity and specificity of immunohistochemical (IHC) detection of HER2 expression is crucial as the role of Trastuzumab have expanded in the treatment of HER2 positive non-breast cancer patients such as gastric cancer patients. Non-specific nuclear and cytoplasmic staining of the HER2 antibody clone 4B5 has been reported by other labs. In this study, we evaluated the specificity of HER2 clone 4B5 and a new HER2 antibody clone UMAB36 using a suite of methods including protein lysate arrays, western blots, FISH and IHC correlation screens on 129 breast and 158 colon cancer cases for HER2 expression. The protein lysate array and western results revealed that clone 4B5 recognizes three proteins: HER2, HER4, and ZSCAN18, a nuclear transcription protein. In comparison, clone UMAB36 recognized only HER2 protein in the same protein lysate array and western screen as clone 4B5. False negative results, based on correlation of IHC with FISH HER2 positives, were generated using clone 4B5 in 1 breast and 5 gastric of the total 287 cancer cases screened. Comparatively, no false negative results were observed using clone UMAB36 in which there was a 100% correlation between IHC and FISH screen. In this study, areas of normal gastric tissue often stained positive by HER2 clone 4B5, so we performed further analysis of 470 normal gastric cases using Ventana9s BenchMark instrument. The results show that 278 normal gastric cases had positive stain with clone 4B5 compared to 3 cases with clone UMAB36. The high background by clone 4B5 may be due HER4 being upregulated in adjacent normal gastric tissue. Our results indicate clone UMAB36 has higher specificity and sensitivity than clone 4B5 in screening gastric tumors. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Ki, Min Zhou, Wei Fu, Xiaozheng Huang, Rachel M. Gonzalez, Youmin Shu, Yi Shen, Guangli Wang, Donghui Ma, Wei-Wu He, Jian Chen. A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein. [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 3921.

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 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