EP-3978525-B1 - MATERIALS AND METHODS FOR PERFORMING HISTOCHEMICAL ASSAYS FOR HUMAN PRO-EPIREGULIN AND AMPHIREGULIN

Inventors

• COUTO, Fernando
• LIAO, ZHIMING
• MURANYI, Andrea
• SHANMUGAM, KANDAVEL
• SINGH, SHALINI
• ZHU, YIFEI

Dates

Publication Date

20260513

Application Date

20160627

Claims (16)

1. An isolated antibody that specifically binds human pro-epiregulin, wherein the antibody comprises the following HVRs: (a) an HVR-H1 comprising SEQ ID NO: 18; (b) an HVR-H2 comprising SEQ ID NO: 19; (c) an HVR-H3 comprising SEQ ID NO: 20; (d) an HVR-L1 comprising SEQ ID NO: 25 or SEQ ID NO: 26; (e) an HVR-L2 comprising SEQ ID NO: 27; and (f) an HVR-L3 comprising SEQ ID NO: 28.
2. The antibody of claim 2, wherein the anitbody further comprises the following heavy chain variable domain and light chain variable domain FRs: (a) FR-H1 comprising SEQ ID NO: 21; (b) FR-H2 comprising SEQ ID NO: 22; (c) FR-H3 comprising SEQ ID NO: 23; (d) FR-H4 comprising SEQ ID NO: 24; (e) FR-L1 comprising SEQ ID NO: 29; (f) FR-L2 comprising SEQ ID NO: 30; (g) FR-L3 comprising SEQ ID NO: 31; and (h) FR-L4 comprising SEQ ID NO: 32.
3. The antibody of claim 1 or 2, wherein the antibody comprises a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO: 34 or SEQ ID NO: 35.
4. The antibody of any one of claims 1-3, wherein the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is a rabbit monoclonal antibody.
5. The antibody of any one of claims 1-4, wherein the antibody is an IgG antibody, or wherein the antibody is an antibody fragment that specifically binds human pro-epiregulin, wherein the antibody fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, and diabody.
6. An isolated nucleic acid encoding an isolated antibody of any one of claims 1-5.
7. A vector comprising the nucleic acid of claim 6.
8. A host cell comprising the vector of claim 7.
9. An immunoconjugate comprising the antibody of any one of claims 1-5.
10. A method of detecting the presence or expression level of human pro-epiregulin in a biological sample in vitro comprising contacting the biological sample with the antibody of any one of claims 1-5 and detecting the presence of the bound antibody.
11. The method of claim 10, wherein the detecting is by immunohistochemistry, immunofluorescence, or immunoblot.
12. The method of claims 10 or 11, wherein the biological sample comprises a fixed tissue.
13. The method of claims 12, wherein the fixed tissue is a formalin-fixed paraffin-embedded (FFPE) tissue.
14. The method of any one of claims 10-13, wherein the biological sample is from a subject having or predisposed to cancer or autoimmune disease, optionally wherein the cancer is colon cancer, breast cancer, or lung cancer.
15. A dispenser for an automated slide stainer comprising a solution comprising the antibody of any of claims 1-5.
16. A kit comprising an antibody according to any of claims 1-5 or a dispenser according to claim 15.

Description

FIELD OF THE INVENTION The present invention relates to antibodies for detecting epiregulin and amphiregulin in human samples and methods of using the same. BACKGROUND About 20% of patients with colon cancer present with metastatic colorectal cancer (mCRC) but regardless of the treatment they receive more than half (50-60%) of these patients will eventually develop incurable advanced disease, which has a 5 year survival rate of approximately 12.5%. Two signaling pathways in mCRC have been the focus of therapeutic drug development: the vascular endothelial growth factor receptor (VEGFR) and the epidermal growth factor receptor (EGFR) pathways. Currently, the majority of the patients with mCRC receive cytotoxic chemotherapy combined with either EGFR or VEGF-targeted therapies. EGFR is overexpressed in about 70% of CRC cases where it is associated with poor outcome. Targeted inhibition of EGFR with monoclonal antibodies, cetuximab or panitumumab, was approved by FDA in 2004 and 2006 to treat patients with mCRC. These antibodies target the extracellular domain of EGFR and compete with endogenous ligands to prevent activation of the receptor. By inhibiting EGFR signaling pathway these biological agents inhibit cell proliferation, differentiation, migration and metastasis. Both drugs have very similar efficacy with a 10-15% response rate. Several molecular markers have been investigated to better predict response to anti-EGFR therapy. See Perkins et al., Pharmacogenetics, Vol. 15, Issue 7, pp. 1043-52 (2014). Clinical studies have provided evidence that EGFR inhibitors are the most effective in patients lacking RAS pathway mutations and maybe detrimental to those who have mutant type tumor. Point mutations in members of the RAS signaling pathways such as KRAS, NRAS, or BRAF lead to continuous activation downstream RAS-MAPK signaling, regardless of whether the EGFR is pharmacologically inactivated. In addition to RAS and BRAF mutations, other alternative mechanisms such as cMET or EGFR amplification play a role in resistance to Cetuximab or Panitumumab. PI3K-AKT-PTEN pathway can also be triggered by EGFR activation therefore mutation in PI3K or PTEN loss (often occur with KRAS or BRAF mutations) is also associated with a lack of response. RAS, BRAF, and PI3K mutations account for more than 60% of patients with mCRC that show de novo resistance to EGFR-targeted monoclonal antibodies. Of the 40% of patients with KRAS, NRAS, BRAF and PI3K wild type tumors (quadruple wild type patients), approximately half of these patients (only 15%) have a major benefit from anti-EGFR therapy and more than 20% are non-responders. Since RAS, RAF, PI3K status is not sufficient to evaluate anti-EGFR response; there is an unmet medical need to improve patients' selection for anti-EGFR therapy. Several potential candidates are under investigations that are involved either in EGFR signaling pathway or in other pathways as MET or HER receptors. Elevated gene expression of epiregulin (EREG) and/or amphiregulin (AREG), ligands for EGFR has been consistently proposed for prediction of anti-EGFR therapy. In these tumors, anti-EGFR antibodies are competing with ligand-dependent activation of EGFR, leading to down regulation of the receptor from the cell surface, thus suppressing proliferative signaling. One recently published study showed that patients whose tumors had low EREG mRNA levels had no benefit from anti-EGFR therapy; the cetuximab therapy was not associated with an improvement in overall survival (OS). While in the biomarker positive group (KRAS wt / EREG high mRNA) the increased EREG mRNA expression was strongly associated with increased therapeutic benefit from cetuximab. In terms of absolute median OS gain, the addition of anti-EGFR therapy increased survival from 5.1 to 9.8 months compared to the best supportive care alone. This result suggests that EGFR ligands expression might become a clinically useful biomarker to screen patients with mCRC for EGFR inhibitor therapy. It therefore would be useful to have new antibodies available for detection of EGFR ligands, such as EREG and AREG, in tissue samples. Pradhan et al. (Nucleic Acids Res. 2014 Jun;42(10):6243-55 ) disclose the use of a polyclonal rabbit antibody against human EREG, termed ab175118. The antibody was raised against a peptide within residues 140-169 of human EREG, and can be applied in Western blotting, immunohistochemistry and flow cytometry. Cell Signaling Technology (http://media.cellsignal.com/pdf/12048.pdf) discloses a rabbit monoclonal antibody recognizing pro-epiregulin and not recognizing the mature form of epiregulin. The antibody was produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu155 of human epiregulin. Zhang et al. (Cancer Prev Res (Phila). 2008 Aug;1(3):201-7 ) disclose the use of a polyclonal goat anti human EREG antibody in immunohistochemistry of primary cancer specimens, wherein the intratumoral epire