Licia Rivoltini, MD Unit of Immunotherapy of Human Tumors

The complex network of anti-tumor immunity Innate immunity First line defense Tumor cell Adaptive immunity Specificity & memory Kidd et al., Nature Biotech 2015

CD8+ T cells: key players in tumor rejection B cell depleted CD8+ T cell depleted Adoptive immune cell transfer CD8+ T cell depleted CD8+ T cells B cell depleted CD4+ T cells NK cells B cells Fox et al., J clin Invest 2015

CD8+ T cells: clonal expansion and immune memory Tumor (?) Expansion of tumor-specific effector cells (optimal priming, adequate cytokines and growth factors contexture) Induction of immune memory (central and effector memory cells for long-term protection)

CD8+ T cells: selective and potent tumor killers TCR Cytotoxic granules (perforin, granzyme B) HLA/Antigen complex Cell apoptosis FasL Fas Immunological synapse Activated CD8+ cytotoxic T cells (CTL) Tumor cell

Homing of cytotoxic CD8+ T cells to tumor site Tumor killing A Initial tumor elimination by NK cells T cell exhaustion Tumor antigens A A A A A Lymph node A A Dendritic cells Process and present Ag to specific T cells Clonal expansion of cytotoxic antitumor CD8+ T cells generation of memory cells Peripheral blood

Immune checkpoints Stop of proliferation Reduced glucose consumption Inhibition of cytotoxicity and cytokine release Blocking of antibody production In a reversible fashion

Immune checkpoint (CTLA4, PD1) anti-pdl-1 Nguyen et al., Nature Rev Immunol 2015

Immune checkpoint (CTLA4, PD1) pakt/pi3k anti-pdl-1 Nguyen et al., Nature Rev Immunol 2015

Predictive factors of response? 30-40% 15% Patrick A. Ott et al. Clin Cancer Res 2013;19:5300-5309

T cell Tumor cell

T cell Neo antigen level of tumor antigens Tumor cell

T cell level of T cell infiltrate level of PD-1 expression in T cells PD-1 Neo antigen level of tumor antigens Tumor cell

T cell level of T cell infiltrate level of PD-1 expression in T cells level of PD-L1 expression in tumor cells (and stroma) PD-1 PDL-1 Neo antigen level of tumor antigens Tumor cell

Tissu antigens Muc1,MART-1, PSMA..) Unique mutated antigens or NEO-ANTIGENS (non-synonimous mutations due to genetic instability)

Tissu antigens Muc1,MART-1, PSMA..) Unique mutated antigens or NEO-ANTIGENS (non-synonimous mutations due to genetic instability) NSCLC Melanoma High mutations High mutations Low mutations Low mutations Lysis of: autologous tumor, common tumors same histotye, common tumor different histotype Rizvi et al. Science, 2015 McGranahan et a., Science 2016

DNA damage response gene mutations, mutational load, and sensitivity to chemotherapy plus immune checkpoint blockade in urothelial cancer DDR mutant UC is associated with Higher mutational load Higher CD8+ T cell infiltrate Potentially higher sensitivity to CT+ICI

T cell level of T cell infiltrate level of PD-1 expression in T cells level of PD-L1 expression in tumor cells (and stroma) PD-1 PDL-1 Neo antigen level of tumor antigens Tumor cell

ADAPTIVE IMMUNE RESPONSE (activated CD8+ T cells) PD-L1 in tumor cells Inducible expression due to local inflammation cytokines and chemokines IFN produced by T cells

PD-L1 in LN, blood and BM ADAPTIVE IMMUNE RESPONSE (activated CD8+ T cells) PD-L1 in tumor infiltrating stroma (myeloid) cells Inducible expression due to local inflammation cytokines and chemokines IFN produced by T cells PD-L1 in tumor cells

PD-L1 in LN, blood and BM ADAPTIVE IMMUNE RESPONSE (activated CD8+ T cells) Surrogated by CD80 PD-L1 in tumor infiltrating stroma (myeloid) cells Inducible expression due to local inflammation cytokines and chemokines IFN produced by T cells Constitutive expression due to oncogenic pathways HIF1a-hypoxia VHL EGFR activation AP-1 signaling PTEN loss PI3K/AKT/mTOR PD-L1 in tumor cells

Impact of immune checkpoint protein expression in tumor cells nd tumor infiltrating CD8+ T cells on clinical benefit from PD-1 blockade in mrcc Expression of either PDL-1 or PD-L2 on tumor cells and low infiltration by T cells expressing multiple IC are associated with better response to ICI

hot hot PD-1 blockade HOT TUMORS COLD TUMORS cold Lizotte et al, J Clin Invest 2016 Daud et al, J Clin Invest 2016

T cell LOW level of T cell infiltrate LOW level of PD-1 in T cells LOW level of PD-L1 in tumor cells (and stroma) LOW level of tumor antigens (neo-antigens) Tumor cell

T cell Immunosuppressive immune components LOW level of T cell infiltrate Myeloid-derived suppressor cells LOW level of PD-1 in T cells LOW level of PD-L1 in tumor cells (and stroma) Tumor associated macrophages Regulatory T cells LOW level of tumor antigens (neo-antigens) Tumor cell

Myeloid derived suppressor cells Granulocytic MDSC (activated neutrophil-like cells) CD33+CD15+CD11b+LDG STAT ROS Monocytic MDSC (immature monocyte-like cells) CD33+CD11b+CD14+HLA-DRneg Arginase-1 TGFb inos PD-L1

Myeloid derived suppressor cells Granulocytic MDSC (activated neutrophil-like cells) CD33+CD15+CD11b+LDG STAT Monocytic MDSC (immature monocyte-like cells) CD33+CD11b+CD14+HLA-DRneg ROS Local effect Arginase-1 TGFb PD-L1 inos Systemic effect Immune suppression Stroma remodelling Neoangiogenesis Mesenchymal transition Pre-metastatic niche Bone metastases

An immunosuppressive protumoral microenviroment defines intrinsic resistance to anti-pd-1 therapy Genes involved in mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, wound healing and myeloid derived suppressor cells (IPRES signature) Hugo et al., Cell 2016

Myeloid Index Score Monocytic MDCS Rivoltini et al., manuscript in prep Sade-Feldman et al., Clin Cancer Res 2016

Absolute neutrophil count Myeloid Index Score Monocytic MDCS Neutrophil to lymphocyte ratio Rivoltini et al., manuscript in prep Sade-Feldman et al., Clin Cancer Res 2016 Ferrucci et al., Ann Oncol 2015

Neutrophil lymphocyte ratio (NLR) as a clinical biomarker predictive of outcomes with immune checkpoint inhibition therapy in genitourinary cancers

Evaluation of monocytic myeloid-derived suppressor cell (m-mdsc) frequency in patients with metastatic urothelial carcinoma

How to improve efficacy? 30-40% 15% Patrick A. Ott et al. Clin Cancer Res 2013;19:5300-5309

Mechanisms of tumor cell death in local therapies Cryoablation Cryoablation Radiotherapy Radioembolization Chemoembolization Cryoablation Radiotherapy Radioembolization Chemoembolization Katrina F. Chu & Damian E. Dupuy Nature Rev Cancer 2014

(presurgery) Ablative therapy to induce local and systemic immune changes in patients with metastatic RCC Intratumor: Increase of PD1+ T cells Higher expression of immune gene signatures Blood: Decrease of Treg cells and IL-6 levels (transient window) Clinical pilot study of anti-ctla4 +/- cryoablation in mrcc patients HCC patients treated with TARE (transarterial radio-embolization) display a window of systemic immune modulation (Rivoltini and Mazzaferro, in preparation)

Pembrolizumab plus low dose ipilimumab for patients with advanced renal carcinoma: phase I KEYNOTE-029 study Managble toxicity and significant activity (disease control rate 50%) Choueiri et al. Evaluation of clinical activity of IPI and NIVO in patients with NIVO-refractory metastatic urothelial cancer IPI and NIVO is active in UC patients who failed NIVO Callahan et al. Cell transition and DNA replication Cell cycle CDK cyclins Differentiation and expansion of T cells Effector T cells NK cell function IL-12/IFNgamma response Differentiation and clonal expansion of CD8+ T cells Granzyme B/perforin lytic machinery Pei-Ling Chen et al. Cancer Discov 2016;6:827-837

Distinct but synergistic effects of CTLA-4 and PD-1 blockade Lymph node Tumor lysis by innate cells (or cancer therapies) and antigen relase Antigen up-take, process and presentation by dendritic cells Priming of antigen-specific T cells by dendritic cells CTLA-4 Effect of blockade Resques dendritic cell tolerance Improves T cell priming by dendritic cells Reduces immunosuppression by Treg Increases T cell proliferation and function T cell clonal expansion and trafficking from regional lymph nodes to tumor site Tumor infiiltration by specific T cells Potentiates T cell effector functions Increases T cell proliferation Rescuses T cells from inhibition by PD-L1+ myeloid-derived suppressive cells Effect of blockade PD-1 Tumor lysis by xx tumor infiltrating T cells Tumor site

Outcomes of PD-1/PD-L1 responders who discontinued therapy for immune related adverse events (iraes): results of a cohort of patients with mrcc In 9 patients, long-term responses after only a median duration therapy of 5 mo (4-15). Induction of Immune memory? Chronic immune stimulation induces memory responses that are blocked by PD-1 a-pd-1 control Im et al., Nature 2016 Utzschneider et al., Immunity 2016

Impact of antibiotics on outcome in patients with mrcc treated with ICI Antibiotic therapy before or during ICI is associated with poor PFS and OS during ICI therapy (n=80)

Impact of antibiotics on outcome in patients with mrcc treated with ICI Antibiotic therapy before or during ICI is associated with poor PFS and OS during ICI therapy (n=80) ICI responding patients expand a microbiota cluster Transplant of the C cluster reverts ICI resistance in mice Vétizou et al,science 2015

To summarize o Strong scientific rational of GU tumors sensitivity to Immune checkpoint inhibitors o Immunogenic tumors (more neoantigens, more mutations, more mutational load, more infiltrating T cells, more PD-1/PDL-1, 2 expression) are more likely to respond o Myeloid-enriched tumors (microenvironmentally or systemically) are more likely to fail responding to ICI(preconditioning required?) o Combinations (ablation, multiple ICI ) should be more effective. But avoid antibiotics (?) o Potential long-term immune memory with PD-1 blockade (like with a-ctla4)

Thank you

Mutation burden as a biomarker of response to immune checkpoint in nine solid cancers: the new concept of icam icam: mutation burden threshold, a gene expression signature of a blocked immune response of robust CD8+ T cell response and up-regulation of immune checkpoints Using data from 10,745 tumors from 33 solid cancer type in TGCA, icam is expressed in Melanoma Lung cancer Colon cancer Endometrial Gastric adenocarcinoma Serous ovarian Bladder urothelial Cervical Her2+ breast cancer icam represents a clinically useful mutation burden threshold