Tyler J. Curiel, MD, MPH curielt@uthscsa Professor of Medicine UT Health Science Center

1. Reversing Immune Dysfunction in Cancer Tyler J. Curiel, MD, MPH curielt@uthscsa.edu Professor of Medicine UT Health Science Center San Antonio, TX

2. Outline • Introduction to tumor immunity • Limitations of the prevailing cancer drug development approach • Failures of the prevailing tumor immunotherapy strategies • The new immunotherapy paradigm and its translational predictions and approaches

3. Louis Pasteur 1822-1895 Germ theory of immunity 1878 First demonstration of acquired immunity with chicken cholera 1880

4. Immune surveillance and tumors Increased cancer in immunosuppressed hosts Spontaneous cancer remissions, especially in renal cell carcinoma and melanoma Demonstration of tumor-specific immunity J Nat CA Inst 1957;18:769 Tumors express antigens Nature 304, 165-7 (1983)

5. ● Is there definitive proof of naturally-occurring immunity against cancers?● Could immune therapy for cancer (of any kind) ever work? The overarching questions ● For which cancers? At what stages? ● What approaches will work?

6. Tumor Immune Surveillance Exists.Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, Schreiber RD IFN-γ and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature. 2001 410(6832):1107-11 Punch Line: T cells, IFN-γ and adaptive (antigen specific) immunity are key elements in defense against tumors

7. Current tumor immunotherapy paradigms build on infectious disease principles that may not apply to cancer T. Curiel J Clin Invest, 117(5):1167-1174 2007

8. One answer: give more T cells Rosenberg, S.A., Spiess, P. & Lafreniere, R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318-21 (1986). LAK cells.Rosenberg, S.A. et al. N Engl J Med 316, 889-897(1987) Morgan, R.A., et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science (2006).

9. Nature Medicine 1996 2(1):52-58 F. Hsu, et al. B-cell lymphoma, autologous antigen-pulsed dendritic cells Nature Medicine 1998 4(3):328 F. Nestle, et al. Melanoma, peptide- or tumor lysate-pulsed dendritic cells

10. Intrinsic tumor strategies • Hide the tumor • Reduce class I • Reduce TAA • Defective Ag processing • Reduce co-signaling • Grow in privileged sites • Prevent active immunity • Prevent cell ingress • Promote cell egress • Kill immune cells • Miscellaneous • Resist apoptosis - Alter cell differentiation

11. DC subsets

12. Tumors reprogram dendritic cells to defeat host immunity, not the tumor Zou, Curiel, et al., Nature Medicine 2001; 7(12):1339-1346

13. Tumor plasmacytoid DCgenerate IL-10+ T cells Zou, Curiel, et al., Nature Medicine 2001; 7(12):1339-1346 .

14. Tumor myeloid DC induce IL-10+ T cells through B7-H1 signals Curiel, Zou, et al., Nature Medicine 2003; 9(5):562-567 VEGF and IL-10 from the tumor induce B7-H1 expression

15. Immune recognition of tumor antigens as self is a significant problem. Infection:rapidly dividing cells of external origin. Cancer: rapidly dividing cells of internal origin. The tumor is a part of the host (self).

16. The big problem • Anti-tumor immunity is autoimmunity. • To generate significant anti-tumor immunity requires breaking self tolerance.

17. Self-reactive Blood, LN, BM, spleen Peripheral tolerance Thymus Negative selection Central tolerance CD4+CD25+ Treg Naïve thymocytes Normal repertoire

18. Treg depletion improves endogenous immunity Shimizu, J., et al. J Immunol163, 5211-8 (1999) Regulatory T cells (Tregs) are CD4+CD25hi T cells Treg depletion improves actively-induced immunity Steitz, J., et al. Cancer Res61, 8643-6 (2001) Sutmuller, et al. J Exp Med194, 823-32 (2001)

19. In tumors, many pathways generate Tregs T. J. Curiel 2007 J Clin Invest 117(5):1167-1174

20. Six fundamental hallmarks of cancerHanahan and Weinberg 2000. Cell 100:57-70 Self-sufficiency in growth signals Evading apoptosis Insensitivity to anti-growth signals Sustained angiogenesis Limitless replicative potential Tissue invasion and metastasis

21. The seventh fundamental hallmark of cancerDunn, G.P., Old, L.J., and Schreiber, R.D. 2004. Annu Rev Immunol 22:329-360.Zitvogel, L., Tesniere, A., and Kroemer, G. 2006. Nat Rev Immunol 6:715-727.T. J. Curiel. 2007 J Clin Invest, 117(5):1167-1174. Lack of immune rejection Self-sufficiency in growth signals Insensitivity to anti-growth signals Evading apoptosis Sustained angiogenesis Tissue invasion and metastasis Limitless replicative potential

22. FOXP3+ Tregs in tumors Curiel, Zou, et al.Nature Medicine10, 942-949 (2004)

23. 40% 17% Treg - + - + 6 40 8 24 IFN-g Counts 24 17 Annexin-V-APC IL-2 Tumor Tregs allow tumor growth despite otherwise sufficient numbers of functional anti-tumor effectors cells Curiel, Zou, et al.Nature Medicine10, 942-949 (2004) IL-2

24. 40% 17% Treg - + - + 6 40 8 24 IFN-g Counts 24 17 Annexin-V-APC IL-2 Curiel, Zou, et al. 2004 Nature Medicine 10, 942-949 Tumor Tregs allow tumor growth despite otherwise sufficient numbers of functional anti-tumor effector cells IL-2

25. Elevated tumor CD4+CD25+ T cells predict poor survival in ovarian cancer Curiel, Zou , et al.Nature Medicine10, 942-949 (2004) 1.0 Low Treg 66.4 mos High Treg 12.8 mos P<0.0001 0.8 0.6 Survival low Treg 0.4 medium Treg 0.2 high Treg 0.0 0 20 40 60 80 100 Months

26. CD4+CD25+ CTCL cell CD4+CD25+ Treg

28. Denileukin diftitox depletesTregs in cancer patients

29. Denileukin diftitox increases blood IFN-γ-producing T cells in cancer patients

30. Patient 4 • Stage IV (metastatic) ovarian cancer. • First recipient of the dose-escalated 12 µg/kg, with significant immune response. • Because she had measurable disease, she received six additional denileukin diftitox doses to test clinical efficacy.

31. Denileukin diftitox reduces metastatic tumor in treatment-refractory ovarian cancer 4 months

32. Corroborating trials • Ovarian: Barnett, B., Kryczek, I., Cheng, P., Zou, W. & Curiel, T.J. Am J Reprod Immunol54:369-377; 2005 • Renal cell: Dannull, J., et al.The Journal of Clinical Investigation115:3623-3633; 2005 • Melanoma:Mahnke, K., et al.Int J Cancer 120: 2723-33; 2007 • Melanoma: Rasku, M. A, et al.J. Translational Med, 6:12;2008

33. Even when the system works,tumors can develop:“The Three Es of Cancer Immunoediting”R. Schreiber Annu Rev Immunol 33:329 2004 Fig: L. Zitvogel et al., Nature Reviews Immunology 6, 715-727 (October 2006)

34. Salvaging DT failure in ovarian cancer S. Wall, S. Thibodeaux, T. Curiel, et al., in preparation Patient SAOC03

35. Interferon-α improves Treg depletion and DT efficacy in ovarian cancer S. Wall, S. Thibodeaux, T. Curiel, et al., in preparation Patient SAOC03

36. How IFN-α boostsTreg depletion effects • Directly activates CD8+ T cells • Boosts T cell-activating capacity of dendritic cells • Increases T cell trafficking into tumor • Does NOT appear to affect Treg function or regeneration after depletion

37. Special cases • Sex • Age

38. Females respond better to anti-B7-H1 blockade in B16 melanoma a 1400 WT + isotype b 1200 WT + isotype 1000 WT+ B7-H1 800 WT+ B7-H1 Tumor volume (mm3) WT + isotype 80 600 WT + isotype 400 p=0.017 200 60 68.0% WT+ B7-H1 0 0 2 4 6 8 10 12 14 16 Suppression (%) 40 Days post B16 challenge WT+ B7-H1 90.8% c Mouse 1 Mouse 2 Mouse 3 20 WT + isotype 0.25% 0.24% 0.27% 0 1:1 1:0.5 WT + isotype Eff:Treg ratio 0.36% 0.35% 0.29% WT + isotype WT + αB7-H1 0.42% 0.46% 0.38% WT + isotype WT+ B7-H1 WT+ B7-H1 WT + αB7-H1 0.55% 0.52% 0.56% p=0.009 6 Pentamerr p=0.028 CD8 4 Total number of tumor-specific CD8+ cells (105) p=0.013 2 0

39. Sex differences in female Tregs • B7-H1-dependent reduction in Treg function • B7-H1 effects are estrogen-dependent • Functional differences are due to defective mTOR/PTEN signaling • Treg function is rescued with dendritic cell B7-H1 signals, estrogen withdrawal or rapamycin

40. Treg depletion does not work in aged female mice with B16 tumor volume (mm3) PBS young DT PBS aged DT day after challenge

41. Aged female mice have more CD11b+Gr-1+ myeloid suppressors that are more suppressive than young 6 p=0.01 4 CD11b+Gr-1+ cells in spleen (%) 2 0 no tumor no tumor PBS DT PBS DT young aged 100 1:1 ratio of MDSC from Spleen 80 60 suppression by CD11b+Gr-1+ from spleen at 1:1 ratio (%) 40 p=0.10 20 p=0.02 p=0.01 0 no tumor no tumor PBS DT PBS DT young aged

42. Depleting Gr-1+ cells improves tumor immunity and slows B16 in aged females tumor volume (mm3) B p=0.019 control mAb young 4 anti-Gr1 p=0.21 control mAb aged anti-Gr1 3 day after challenge Percent IFNγ+ of CD8+ T cells in spleen 2 1 0 no tumor control mAb -Gr-1 mAb control mAb -Gr-1 mAb young aged

43. Summary and conclusions • Cancers are immunogenic and thus should be amenable to effective immune therapies in the new paradigm. • Immune therapies are adjuncts in multi-modal treatment approaches. • Immune therapy is not appropriate for all patients.

44. Ways forward • Identify patients with relatively intact immune systems for trials • Test available agents: DT, anti-CTLA-4 • Test reversing immune dysfunction with immunization or immune boost (e.g., anti-CTLA-4 or DT plus a vaccine)

45. Final Thoughts • We need a better understanding of immune dysfunction in cancer. • We need a better understanding of the immune effects of current agents. • Willingness of investigators to try immune therapies will help, but they have to be convinced.

46. Acknowledgements • Curiel lab members • National Cancer Institute • Hayes, Voelcker, Rippel Foundations and Trusts, Eisai • UTHSCSA endowments • Cancer Therapy & Research Center