Preclinical rationale for Herceptin treatment beyond progression in HER2-positive breast cancer

1. Preclinical rationale for Herceptin treatment beyond progression in HER2-positive breast cancer

2. Established chemotherapy resistance mechanisms Impaired drug uptake Active drug efflux, eg by ABC transporters (P-glycoprotein, MDR2, BCRP, MRP1-6 etc) Enhanced drug metabolism, eg by P450 enzymes Alterations of intracellular target, eg tubulin Upregulation of DNA repair in tumour cells Upregulation of signalling pathways, eg anti-apoptotic genes (bcl-2, XIAP etc)

3. Hypothetical mechanisms of resistance to Herceptin (1) Selection of HER2-negative cells in a heterogeneous tumour Outgrowth of HER2-negative tumours from an originally mixed tumour cell population Defective interaction of Herceptin with HER2 Masking of Herceptin-binding epitope of HER2 Alterations in Herceptin-binding epitope of HER2 Loss of HER2 ECD by shedding or alternative initiation of translation on HER2 gene 1. Kunitomo et al. Hum Pathol 2004; 35: 379-381 (case report). 2. Nagy et al. Canc Res 2005; 65: 473-482. 3. Tanner et al. Mol Cancer Ther 2004; 3: 1585-1592. 4. Stephens et al. Nature 2004; 431: 525-526. 5. Stephens et al. Nature Genetics 2005; 37: 590-592. 6. Anido et al. EMBO J 2006; 25: 3234-3244.1. Kunitomo et al. Hum Pathol 2004; 35: 379-381 (case report). 2. Nagy et al. Canc Res 2005; 65: 473-482. 3. Tanner et al. Mol Cancer Ther 2004; 3: 1585-1592. 4. Stephens et al. Nature 2004; 431: 525-526. 5. Stephens et al. Nature Genetics 2005; 37: 590-592. 6. Anido et al. EMBO J 2006; 25: 3234-3244.

4. Hypothetical mechanisms of resistance to Herceptin (2) Changes in downstream signalling proteins which eventually disconnect growth regulation from HER2 PIK3CA mutations resulting in constitutively active PI3-kinase Loss of PTEN function leading to persistent signalling activity via the PI3K/Akt survival pathway Changes in cyclin-dependent kinase inhibitor p27kip1 1. Berns et al. Cancer Cell 2007; 12: 395-402. 2. Nagata et al. Cancer Cell 2004; 6: 117-127. 3. Crowder et al. Cancer Cell 2004; 6: 103-104. 4. Pandolfi. N Engl J Med 2004; 351: 2337-2338. 5. Kute et al. Cytometry 2004; 57A: 86-93. 6. Nahta et al. Cancer Res 2004; 64: 3981-3986.1. Berns et al. Cancer Cell 2007; 12: 395-402. 2. Nagata et al. Cancer Cell 2004; 6: 117-127. 3. Crowder et al. Cancer Cell 2004; 6: 103-104. 4. Pandolfi. N Engl J Med 2004; 351: 2337-2338. 5. Kute et al. Cytometry 2004; 57A: 86-93. 6. Nahta et al. Cancer Res 2004; 64: 3981-3986.

5. Hypothetical mechanisms of resistance to Herceptin (3) Engagement of alternative growth factor receptor pathways Autocrine production of EGF-like ligands, eg heregulin Upregulation of HER-family receptors, eg HER3 Upregulation of non-HER-family growth factor receptor-mediated signalling, eg IGF-1R, cMET 1. Motoyama et al. Cancer Res 2002; 62: 3151-3158. 2. Lee-Hoeflich et al. Manuscript submitted to Cancer Res. 3. Lu et al. J Natl Cancer Inst 2001; 93: 1852-1857. 4. Albanell et al. J Natl Cancer Inst 2001; 93: 1830-1832. 5. Lu et al. Int J Cancer 2003; 108: 334-341. 6. Shimizu et al. Mol Pathol 2004; 35: 1537-1542. 7. Altundag et al. Mol Pathol 2005; 36: 448-449.1. Motoyama et al. Cancer Res 2002; 62: 3151-3158. 2. Lee-Hoeflich et al. Manuscript submitted to Cancer Res. 3. Lu et al. J Natl Cancer Inst 2001; 93: 1852-1857. 4. Albanell et al. J Natl Cancer Inst 2001; 93: 1830-1832. 5. Lu et al. Int J Cancer 2003; 108: 334-341. 6. Shimizu et al. Mol Pathol 2004; 35: 1537-1542. 7. Altundag et al. Mol Pathol 2005; 36: 448-449.

6. In vitro studies are not predictive of in vivo resistance In vitro resistance was observed in cell lines exposed to Herceptin In vitro resistance models tend to focus on just one biological feature In vitro resistance represents intrinsic insensitivity or artificial manipulation of cells Conclusions from in vitro resistance models cannot be translated to clinical settings ADCC is a key mechanism of Herceptin efficacy in vivo Gennari R, et al., Clin Cancer Res. 10(17):5650-5, 2004 Arnould L, et al., Br J Cancer 30;94(2):259-67, 2006 Musolino A, et al., J Clin Oncol. 26(11):1789-96. 2008 Gianni L, J Clin Oncol. 26(11):1779-90. 2008 Gennari R, et al., Clin Cancer Res. 10(17):5650-5, 2004 Arnould L, et al., Br J Cancer 30;94(2):259-67, 2006 Musolino A, et al., J Clin Oncol. 26(11):1789-96. 2008 Gianni L, J Clin Oncol. 26(11):1779-90. 2008

7. ADCC is a key mechanism of Herceptin’s antitumour activity in vivo

8. Herceptin inhibits the growth of JIMT-1 xenograft tumours in vivo despite in vitro resistance

10. Herceptin significantly decreased the number of JIMT-1 tumour cells in blood and bone marrow despite progression of the primary tumour Barok et al. Cancer Letters 2008; 260: 198-208.Barok et al. Cancer Letters 2008; 260: 198-208.

11. JIMT-1 cells isolated from Herceptin-treated progressing tumours retain sensitivity to ADCC JIMT-1 cells resistant to both Herceptin and Herceptin F(ab’)2 fragments in vitro are sensitive to Herceptin, but not Herceptin F(ab’)2 when grown as xenografts in vivo Herceptin significantly reduces the number of circulating / disseminated tumour cells in this xenograft model system despite the primary tumour being unresponsive to Herceptin Barok et al. Mol Cancer Ther 2007; 6: 2065-2072. Barok et al. Cancer Letters 2008; 260: 198-208. Barok et al. Mol Cancer Ther 2007; 6: 2065-2072. Barok et al. Cancer Letters 2008; 260: 198-208.

12. Herceptin treatment beyond progression enhances efficacy of combination chemotherapy HER2 remains overexpressed and active in progressive disease HER2 may contribute to an even more aggressive tumour growth if Herceptin treatment is discontinued Inhibition of HER2 signalling may sensitise tumours to chemotherapy in tumours progressing on Herceptin alone

13. Herceptin treatment beyond progression sensitises tumour cells to chemotherapy

14. Herceptin treatment beyond progression enhances efficacy of combination therapy with targeted agents Herceptin synergistically enhances the antitumour effect of Avastin in tumours progressing on Herceptin Herceptin synergistically enhances the antitumour effect of pertuzumab in tumours progressing on Herceptin Lapatinib enhances the antitumour effect of Herceptin

15. Herceptin + Avastin combination eradicates tumours in a breast cancer xenograft model

16. Herceptin + pertuzumab combination eradicates tumours in a breast cancer xenograft model

17. Combination of Herceptin and lapatinib is clearly synergistic M Scaltriti et al. Lapatinib induces HER2 stabilization and enhances the antitumor activity of trastuzumab in vivo. Abstract 2559, AACR 2008. M Scaltriti et al. Lapatinib induces HER2 stabilization and enhances the antitumor activity of trastuzumab in vivo. Abstract 2559, AACR 2008.

18. Conclusions In vitro studies are not predictive of in vivo resistance Herceptin is not subject to classical chemotherapy resistance mechanisms As activation of the immune system (ADCC) is a key mechanism of action for Herceptin, alternative signalling does not lead to in vivo resistance Preclinical data suggest that continuation of Herceptin treatment beyond progression in combination with chemotherapy or targeted agents can lead to tumour eradication