Why did Science rank immunotherapy as #1?

2. Why did Science rank immunotherapy as #1? !!!

3. Specific immunotherapy Active immunotherapy Antibody therapy Adoptive transfer of T cells Vaccine-based immunotherapy Tumour-based vaccines Virus-based vaccines Peptide-based vaccines others Immunotherapy

4. Abridged history of cancer immunotherapy Lesterhuis et al, Nature Reviews 2011 2011: Ipiliumumab shows overall survival benefit in melanoma (2011) 2012-2013: PD1 and PD-L1 blockade has benefit in melanoma, NSCLC, renal cell (4 NEJM papers) 2011-2013: CAR-modified T cells show durable remissions in B cell ALL and CLL

5. Can we combine local RT, antibodies to oncogenic receptors , selected chemo, hormones to alter tumor microenvironment that allow immunotherapy to optimize host immune response for tumor regression? Major barriers in immunotherapy • Difficult to break tolerance • Poor recruitment to tumor site • Strong suppressive environment within the tumor site • Fast growing tumor in mouse model • Lack of defined antigens and adjuvant for CTL

6. T cell based therapy

7. Human CD19 expression by hematopoietic cells within the spleen and lymphoid tissues Tedder, T. F. (2009) CD19: a promising B cell target for rheumatoid arthritis Nat. Rev. Rheumatol. doi:10.1038/nrrheum.2009.184

8. Using CAR to Overcome Tolerance Creation of Bi-specific T cells TCR heterodimer approach “CAR” or T body approach TCR Chimeric Protein - off the shelf - MHC independent   Tumor binding domain CD3 Extracellular Intracellular Signaling domain        - ITAM

9. Design of CART19: Choice of 4-1BB Signaling Domain Promotes CAR T Cell Proliferation/Survival

10. New strategies that target tumor microenvironment and generate local and systemic immune protection

11. TUMOR Lead Cover Lead Cover Lead Cover Improved Targeting of X-rays tumor Lead Plate X-ray Transparent Plexiglass X-Rays 11 Backscatter Lead Cover

12. Nude Wt 7500 6000 Control Control 6000 RT RT 4000 4500 Tumor volume (mm3) Tumor volume (mm3) 3000 2000 1500 0 0 0 10 20 30 40 0 10 20 30 40 Days post RT Days post RT *** Local Ablative RT mediates tumor regression B16-SIY: 20Gy or B16: 20Gy x 2 die RT-mediated tumor regression depends on T cells Traditional low dose and hyper-fractionated RT can not control tumor Cure all die Blood 2009, Cancer Res. 2011, JI 2013, JCI in press

13. 100 Control 75 a RT+ CD8 Percent survival RT 50 25 0 0 10 20 30 40 50 60 70 Days post tumor injection Which immune cells controls tumor growth CD8+ T cells are required for therapeutic response to RTIncrease of PDL-1 induces resistance and allows relapse B16 RT on day 14: 15Gy x day 14, 15, ad 16 2000 *** ) 3 Control 1500 a RT+ CD8 RT 1000 Tumor volume (mm 500 ** 0 0 3 6 9 12 15 Days post RT

14. 25000 *** 20000 cpm 15000 10000 5000 0 RT-DC+T RT-DC only No RT-DC+T No RT-DC only Which cytokines? can RT increase cross-priming of tumor Ag by intratumor DCs? B16-SIY 20 Gy 14 days 3 days CD11c+ from tumor 2C T cells Proliferation Assay

15. Why is cross-priming increased • It is known that IFNs can be induced by viral DNA for cross priming of CTL. • Hypothesis: RT-induce DNA damage leads to excessive DNA fragments, like viral infection, which trigger IFNs that induce DC maturation and cross priming • RT induces IFN inside tumor tissues

16. 5000 IFNAR KO Control ) IFNAR KO RT 3 4000 WT Control WT RT 3000 Tumor Volume (mm 2000 1000 ** 0 0 5 10 15 Days post RT The therapeutic response to RT is dependent on type I IFNs RT induced tumor regression depends on STING but not MyD88 and TRIF While anti-Her2/neu uses MyD88.

17. RT and anti-PD-L1 reduces MDSC through CTL harvest A Ctrl RT+αPD-L1 RT+αPD-L1+αCD8 19.9% 0.55% 10.2% Gr-1 CD11b Re-activated CTL kills Ag+ cells Including MDSC and tumor B RT TUMOR αB7-H1 mAb (but not anti-PD-1) Day 0 14 17 20

18. anti-HER2/neu antibody reduces tumor burden: how? Oncogenic blockade FcR mediated kill? NK TUMOR TUMOR

19. WT mIgG Wt -neu Rag-/- mIgG Rag-/--neu Tumor model that depends on oncogenic signals and FcR dependent in immunocompetent host 1250 TUBO derived from MMTV-Rat neu Tg FcR and NK dep. 1000 750 Tumor volume (mm3) 500 ** Rag-1 +Ab Wt + Ab 250 * 0 0 10 20 30 40 50 Days after tumor inoculation Are T cells essential for Ab-mediated tumor regression? Cancer Cell, 2010

20. Ab-mediated tumor regression is CD8 dependent: wt and Tg mice A) B) Ctrl Ctrl -neu -neu Neu Tg mice -neu + -CD8 -neu + -CD8 Tumor volume(mm3) Tumor volume(mm3) ** * * Naive 2000 -neu 1500 Days after tumor inoculation Days after tumor inoculation 1250 1000 700 1000 1250 600 C) D) 500 750 500 1000 ** Ctrl 400 0 500 750 -neu 3T3 KB TUBO 300 250 IFN + reactive cells / 106 splenocytes 200 500 memory Tumor volume (mm3) 100 0 250 0 10 20 30 40 50 0 0 10 20 30 40 50 60 0 0 10 20 30 40 50 60 70 80 Days after tumor re-challenge Cancer Cell, 2010, MT, 2013, CCR 2013

21. How can antibody trigger immune responses? CD8 IFN cytokines Increased Cross-priming ADCC Stress protein or DNA TLRs? MyD88

22. Type I interferons are induced and necessary during antibody-mediated tumor regression. A TUBO Ab or adenovirus-IFN * WT mice * 0 1 2 3 4 5 6 wk B Ab can induce IFN for tumor regression but antibody can not induce IFN in Ab-resistant tumor Could exogenous IFN be potent for targeting tumor? *

23. How to target tumor with IFN: armed Ab with IFN for Ab-resistant tumor B16-EGFR 25ug Ab or Ab-IFN WT mice 0 1 2 3 4 5 6 wk Anti-EGFR Linker IFNb Anti-EGFR-IFNb is effective for controlling Ab resistant tumor