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Lin Nie, Olga Burenkova, Stavros Kopsiaftis, Sharlene Adams, Ashish Kalra, Aaron Fulgham, Dongmei Xiao, Viara Grantcharova, Matt Onsum, Sharon Moulis, Lucia Wille, Gabriela Garcia, Victor Moyo, Bill Kubasek, Ulrik NielsenMerrimack Pharmaceuticals, Inc., 700 One Kendall Sq, Cambridge, MA 02139

Efficacy of MM-121 in ER+ and triple negative breast cancer studies #1806

1) BackgroundIn women, breast cancer is among the most common cancers and the fifth most common cause of cancer deaths. Due to the heterogeneity of the disease, 10-year progression free survival can vary widely with stage and type from 98% to 10%. We present data showing that MM-121, a fully human monoclonal anti-ErbB3 antibody, is efficacious in studies of both hormone dependent (ER+) and triple negative (ER-,PR-, Erb2 low) breast cancer lines that express the molecular profile consistent with MM-121 response (co-expression of ErbB3 and HRG). . Using a combination of computational and experimental approaches, ErbB3 was identified as a critical transducer of oncogenic signaling (Fig 1) leading to the development of MM-121, a first in class anti-ErbB3 antibody. We have previously demonstrated that MM-121, when used as a single agent, inhibits heregulin-induced signaling events in human

2) Identification of a MM-121 positive biomarker signature in multiple breast cancer cell lines

3) MM-121 treatment inhibits VEGF secretion in breast cancer cell

4) Treatment with MM-121 inhibits proliferation and induces apoptosis in vitro

6) MM-121 in combination with Tamoxifen inhibits tumor growth greater than either single agent alone

7) MM-121 in combination with paclitaxel inhibits cell growth greater than either single agent alone

8) Observations

• Multiple breast cancer cell lines have an MM-121 positive biomarker signature. • In vitro, MM-121 induces cell cycle arrest, apoptosis and inhibits VEGF

production in various human breast cancer cell lines.• MM-121 monotherapy is efficacious in both ER+ positive and triple negative

breast cancer xenografts.• In BT474-M3 cells tamoxifen treatment results in a change in the MM-121

biomarker signature in vivo. In BT474-M3 xenograft studies, combination treatment shows tumor growth inhibition greater than either single agent alone.

• The MM-121 plus paclitaxel combination is effective in vivo in both ER+ and triple negative breast cancer cell line.

• MM-121 has potential as a single agent or in combination with other therapies in ER+ and TNBC.

A) MM-121 can inhibit HRG dependent VEGF secretion. MCF-7 and T47D cells (0% FBS) were treated with either HRG alone (10nM for 48hrs) or HRG in combination with MM-121 (2uM, 1hr pre-treatment) and VEGF protein levels were measured in culture supernatant by ELISA (R&D). B) MM-121 can inhibit endogenous VEGF secretion. MCF-7 and T47D cells were treated with MM-121 (2uM, 48hrs). VEGF protein levels were measured in culture supernatant by ELISA (R&D). C) MM-121 can inhibit both endogenous and HRG induced VEGFa mRNA production in MCF-7 cells. MCF-7, BT474-M3 cells (2%FBS) were treated with MM-121 (500nM, 1hr pre-treatment) +/- HRG (10nM, 5hrs) and VEGFa mRNA levels were measured by RT-PCR.

A) MDA-MB-175-VII cell viability is inhibited by MM-121. MDA-MB-175-VII cells (20% FBS) were treated with MM-121 for 6 days and cell viability was assayed by cell titer glo (Promega). B) MM-121 induces cell cycle arrest in MDA-MB-175-VII cells. Cells (20% FBS) were treated with MM-121 for 96 hrs. then trypsinized (0.05%) and stained using Propidium Iodide. FlowJo software was used for analysis. C) MM-121 induces apoptosis in MDA-MB-175-VII cells. Cells (20% FBS) were treated with MM-121 for 48 hrs. then trypsinized (0.05%) and stained using Propidium Iodide and Annexin V. D) MCF-7 cells upregulate Bax in response to MM-121 treatment. MCF-7 cells (10% FBS) were treated with MM-121 for 48hr, lysted and Western blots were probed for Bcl-2 (Santa Cruz) and BAX (Santa Cruz).

A) Tamoxifen treatment reduces pErbB3 and total ErbB3 levels in BT474-M3 cells. Western blots of BT474-M3 tumors lysates (T-PER, Thermo Scientific) were probed with antibodies against phospho-ErbB1 (Tyr1173), ErbB3, phospho-ErbB3 (Tyr1289), phospho-Akt (Ser473) and beta-actin (all antibodies were purchased from Cell Signaling Technologies). B) BT474-M3 xenograft tumors. Nu/nu mice were implanted with estradiol pellets (0.72mg/pellet/60days, Innovative Research of America) and 19x106 BT474-M3 cells/mouse. 7 days post implantation, mice were randomized based on tumor volume and treated with MM-121 (Q3D) and/or tamoxifen (pellet 5mg/pellet/60days, Innovative Research of America)

A) MDA-MB-175-VII cell viability decreases in vitro in response to MM-121 and paclitaxel combination treatment. MDA-MB-175-VII cells (20% FBS) were treated with MM-121 and/or paclitaxel for 4 days and cell viability was assayed by cell titer glo (Promega). C) MDA-MB-231 xenograft tumors treated with MM-121 in combination with paclitaxel. Balb/c nude mice were implanted orthotopically with 20x106 MDA-MB-231 cells/mouse (low growth factor matrigel, BD Biosciences). Mice were randomized based on tumor volume and treated with MM-121 (Q3D) and/or paclitaxel Q7D (LC labs) C) BT474-M3 xenograft tumors treated with MM-121 in combination with paclitaxel. See adjacent panel for methods.

ER* PR* Her2* Mutation status**

BT474 + + + PI3K wt, p53 E285K(?),

MCF-7 + + PI3K E545K, BRAF wt, PTEN wt, BRCA1 wt, KRAS wt, p53 wt, CDH1 wt

MDA-MB-231 - - KRAS G13D , BRAF G464V, ATM wt, PTEN wt, PI3K wt, p53 R280K, BRCA1 wt, BRCA2 wt, p53 R280K(?), CDH1 wt

T47D + + PI3K H1047R, BRAF wt, KRAS wt, PTEN wt, BRCA1 wt, BRCA2 wt, p53 L194F(?), CDH1 wt

MDA-MB-175-VII + - PI3K wt, BRAF wt, KRAS wt, PTEN wt, p53 wt, CDH1 wt

* = Neve et al., Cancer Cell 10, 2006

** = Sanger Database

A) Predictions of MM-121 responders and non-responders based on biomarker signature. 5 signaling proteins, including receptors and ligands of the ErbB pathway, were assayed to predict MM-121 xenograft response for 7 breast cancer cell lines. For a more detailed explanation see Poster # 3765. Breast cancer cell lines shown in bold. B) Mutation status in select breast cancer cell lines

Figure 1. Schematic of the ErbB pathway. The computational model of ErbB signaling is based on literature supported interactions of the four ErbB receptors. Ligand binding, receptor homo- and heterodimerization, receptor internalization, recycling and degradation are included in the model.

cancer cell lines. Moreover, MM-121 caused dose-dependent inhibition of tumor growth in multiple xenograft models of human cancer, including ovarian, renal cell, pancreatic, lung, and prostate cancer. Here, we show that when MM-121 is used as a single agent or in combination with other therapies, it could offer significant clinical benefit to both ER+ and triple negative breast cancer patients

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MM-121 is efficacious in both cell line and primary human xenografts that are representative of either the ER+ or triple negative breast cancer population.Nu/nu (Charles River lab) mice were inoculated with indicated cell lines (A and B) or implanted with human tumor specimens (Oncotest) (C). Established tumors were dosed every 3 days with either PBS or MM-121 at 600 ug/mouse (B, C) or 1500 ug/mouse (A).

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