Cell adoptive cancer Immunotherapy

SCI. Barcelona, 18/11/2016 Cell adoptive cancer Immunotherapy Daniel Benítez Ribas, PhD Dept of Immunology. Hospital Clínic Barcelona

Breakthrough of the year 2013 Cancer Immunotherapy

Specificity Block inhibitory pathways Tumor-specific T cell recognition in the periphery Lymphocyte priming to tumor antigens Only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade

Tumor immunogenicity WEAK IMMUNOGENIC STRONG IMMUNOGENIC

The cancer immunity cycle

Therapies that might affect the cancer-immunity cycle

Introduction The primary goal of cancer immunotherapy is to activate the immune system in cancer patients Tumors can be immunogenic Recognition of tumor antigens can lead tumor elimination Immunotherapy aiming to stimulate T lymphocytes Antigen (tumor)-specific T lymphocytes eliminate tumor Memory responses minimal residual disease (metastasis)

Tumor antigens Based on their molecular structure and source 1. Products of mutated oncogenes and tumor suppressor genes 2. Product of other mutated genes 3. Over expressed or aberrantly expressed cellular proteins 4. Tumor antigens produced by oncogenic viruses 5. Oncofetal antigens 6. Altered glycolipids and glycoproteins 7. Cell type-specific differentiation antigens

Characteristics of an ideal cancer antigen Therapeutic function Immunogenicity Oncogenicity Specificity Criteria Expression level and % positive cells Stem cell expression Top subcriteria Superb data controlled vaccines trial suggestive T-cell and/or antibody responses elicited in clinical trials Associated with oncogenic process Absolutely specific Highly expressed on all cancer cells in patients designated for treatment Evidence for expression on putative cancer stem cells Nº patients with antigen-positive cancer High level of expression in many patients with a particular tumor type Nº epitopes Longer antigen with multiple epitopes and the potential to bind to most MHC-molecules Cellular location of expression Normally expressed on the cell surface with no or little circulating antigen

Three ways for self antigens to become tumor antigens OJ Finn. Cancer Immunology. N Engl J Med, 2008; 358:2704-15

Immunostimulatory and Immunosuppressive Forces in the Tumor Microenvironment OJ Finn, Cancer Immunology. N Engl J Med, 2008; 358:2704-15

Therapeutic Approaches NATURE OUTLOOK. CANCER IMMUNOTHERAPY. Nature 2013, 504: 7480, S1-S16

Generation/induction antigen (tumor)-specific T cells Indirect (Active immunization) Cancer vaccines (tumor lysates, cells, peptides, DNA, mrna) Dendritic cells (loaded with lysates, proteins, peptides, mrna) Direct (ACT) TIL (Tumor-infiltrating lymphocytes) TCRT (TCR engineered T cells) hatcr (high affinity TCR engineered T cells) CAR-T (Chimeric antigen receptor engineered T cells)

Generation/induction antigen (tumor)-specific T cells Indirect (Active immunization) Cancer vaccines (tumor lysates, cells, peptides, DNA, mrna) Dendritic cells (loaded with lysates, proteins, peptides, mrna)

Cancer vaccine: Heterologous Melanoma cell lines vaccine Whole cell vaccine Therapeutic polivalent vaccine 1) heterogeneous expression of the cell surface antigens (melanomaassociated antigens, common surface antigens and HLA antigens) 2) balanced representatives of primary, lymph node and metastatic melanoma cells 3) heterogeneity with respect to their in vitro growth rate; and 4) absence of viral (HIV, hepatitis and HTLV), bacterial and fungal infectious organisms in the donor serum

Whole cell vaccine Cancer vaccine: Heterologous Melanoma cell line vaccine THERAPEUTIC POLIVALENT VACCINE PREPARATION MELANOMA CELL LINES GENERATION, SELECTION AND EXPANSION (10 cell lines) Discarded microbiological contamination Growing No production of IL-10 and TGF-b, FasL Phenotype Surgical localization MICROBIOLOGICAL TESTING MIXED OF MELANOMA LINES STORING FROZEN AT N 2

Vaccine Administration Whole cell vaccine THAWED IRRADIATION DNFB ADITION OF BCG INTRADERMAL INOCULATION CLOSE TO LYMPH NODES MONTHLY (FIRST YEAR), EVERY THREE MONTHS (SECOND YEAR) AND EVERY SIX MONTHS (THIRD YEAR)

CLINICAL RESULTS Whole cell vaccine PACIENTS AVALUABLE DISEASE: 23 RESPONSES: 6 (26%) 3 COMPLETES (18, 10+, 20+ months) 2 PARTIALS (8, 16+) 1 MIXES (30+) WITHOUT DISEASE: 15 5 (33%) STABLE (Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine. Int J Cancer 2003)

Complete remission of the multiple subcutaneous metastasis after 1.5 year of treatment

Complete remission of the multiple subcutaneous metastasis after 8 months of treatment

Update 10 years extension Stage MM Patients Follow up Median survival Mean survival SD Stage IIIC 23 13 years 22.5 month 30.7 months 27.7 months Stage IV 41 12 years 13.0 months 22.1 months 30.9 months

Conclusions Therapeutic vaccines It is feasible and safe to apply allogeneic tumor cells (irradiated) No side effects, the vaccine is well tolerated Inactivated whole cell vaccines have an impact in clinical responses in melanoma patients (Overall survival and complete responses) How to boost antigen (tumor) presentation?

Cellular response (adaptive) Natural Immune Response

Clinical application of DCs In human Immunogenic DCs Cancer Melanoma Colon Breast Prostate Glioma RCC HIV Tolerogenic DCs Allergy Transplantation Autoimmunity (RA)* Immune-based disorders (IMID)* MS/NMO Diabetis

Dendritic cell based clinical trials (2016 active) Map from clinicaltrials.gov

Why to use cells as therapeutic agents? Therapeutic agents Small molecules Biologics Cells Move to specific sites in the body Cells can sense diverse signals (sense surroundings) Integrate inputs to make decisions Execute complex response behaviors

DC clinical application. General considerations DC (modcs, circulating pdcs, mdcs, CD34 + derived) application in human patients is safe and well tolerated DCs induce Ag-specific T cells and humoral responses modifying the type of cytokines and antibody switching Some patients (tumor) develop efficient clinical responses (room for improvement and optimization)

Ex vivo culture of DCs. Natural DCs or their precursor cells Immature DC Growth factors Maturation factors Tumor antigens CD34+ cells Monocytes Natural DCs Apheresis Administration Clin Cancer Res; 22(8) April 15, 2016

DC-based therapy in stage IV melanoma patients Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell lines lysate CD14 - MHC II + CD40 +/- CD80 +/- CD83 - CD86 + IL-1b, IL-6 TNF-a, PGE 2 Tumor cell lines lysate CD14 - MHC II +++ CD40 + CD80 ++ CD83 + CD86 +++

DC-based therapy Clinical results 11 patients stage IV MM (post-biochemotherapy) 1 PR (partial response lasting 5 months) 2 MR 8 PD dendritic cell therapy: is well tolerated, induces an immune response shows promising antitumoral activity in stage IV melanoma patients Cancer Immunol Immunother. 2004 Jul;53(7):651-8

DC vaccination: status After 20 years of DC vaccination: We can now induce an immune response in 40% of stage IV melanoma patients Patients with immune responses show increased progression free survival, but long-term clinical responses are still limited (25%) Objective clinical responses (5-10%) Monocyte derived DC vaccines are not yet optimal: limited survival, migration, co-stimulation, activation, Ag presentation, DC subsets Most patients treated are late stage cancer patients (2-3 line)

Dendritic cells in cancer immunotherapy Clinical Response Overall Survival Melanoma 8.5 % 50 % - 377 % Prostate Cancer 7.1 % 56 % - 175 % Malignant Glioma 15.6 % 49 % - 150 % Renal Carcinoma 11.5 % 108 % More than 3000 patients have been treated with DCs Data extracted from: Clinical use of dendritic cells for cancer therapy. Lancet Oncology 2014 Sébastien Anguille, Evelien L Smits, Eva Lion, Viggo F van Tendeloo, Zwi N Berneman

Clinical response and DTH Skin-derived specific T cells DTH 6 mm Biopsies 100 Stage III melanoma patients 100 Stage IV melanoma patients 80 80 percentage 60 40 specific T cells (n=24) percentage 60 40 specific T cells (n=8) 20 20 0 0 10 no specific T cells (n=7) 20 30 40 50 60 0 0 10 no specific T cells (n=18) 20 30 40 50 60 Progression free survival (months) Progression free survival (months) De Vries et al. J Clin Oncol 2005

Dendritic Cell Immunotherapy: Mapping the way Figdor et al Nature Med. 2004

Plasmacytoid DC are major type I IFN producers BDCA-2 Plasmacytoid DC Plasmacytoid DC are antigen-presenting cells. scarce (less than 0,1% of peripheral blood leukocytes). the major type I IFN producers. critical for anti-viral immunity. not yet well understood. CD123

DC vaccination: pdcs? Immature pdcs infiltrate solid tumors Type I IFN seems to yield more potent DCs in terms of secretion of IL- 12 and induction of tumor-specific CTLs and Th1 in vitro pdcs create the appropriate environment for efficient CTL response against viruses Activated and injected together with mdcs, pdc may improve the antitumor responses (animal models)

pdc: Clinical grade purification Apherese (4h) pdc isolation BDCA-4 (6h) O/N with rhil-3 (10 ng/ml) Quality control: Phenotype Purity Yield Viability TLR-ligand stimulation (6h) Peptide loading (last 2h) Injection (intranodal) Quality control: Phenotype IFN-α production T cell stimulation Prophylactic vaccines mimic synthetic CpG oligonucleotides in their ability to modulate immune responses. Mol Immunol. 2011 Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells. Blood, 2010

Schematic overview of the pdc culture protocol and vaccination strategy. Jurjen Tel et al. Cancer Res 2013;73:1063-1075 2013 by American Association for Cancer Research

Activated pdcs are mature and migrate to distinct lymph nodes in vivo. Jurjen Tel et al. Cancer Res 2013;73:1063-1075 2013 by American Association for Cancer Research

pdcs induce immune responses to FSME in vivo T cell responses FSME specific T cell response 16384 ns Humoral responses FSME - IgG in Blood T cell Proliferation - CPM 8192 4096 2048 1024 512 Arbitrary units (U/ml) 300 200 100 ns *** 256 128 Before vacc. After 3 vacc. 0 Before Vacc. After 1 Vacc. After 2 Vacc. After 3 Vacc. Frühsommer-Meningoenzephalitis (FSME; tick-borne encephalitis) activates pdcs via TLR9 and TLR7

Tumor specific T-cells in 8 vaccinated patients DTH MLPC Tetramer APC gp100 154 gp100 280 tyrosinase gp100 tyrosinase 0.4 % 0.2 % 0.2 % 0.1 % 0.3 % 0.4 % Frequency of antigen specific CD3 + CD8 + T cells 10-5 10-6 10-7 gp100 154 gp100 280 tyrosinase ** ns ns CD8 FITC

pdc vaccination improves OS Clinical outcome to pdc vaccination was compared with a group of carefully matched historical control patients who received dacarbazine as first-line treatment Jurjen Tel et al. Cancer Res 2013;73:1063-1075 2013 by American Association for Cancer Research

Clinical outcome after vaccination with pdc Overall survival stage IV melanoma patients Percent survival 100-80- 60-40- 20- pdc (n=15) P=0.001 1-year survival: - historical matched controls 35 % - pdc group 60% 2-year survival: - historical matched controls 10 % - pdc group 45% matched controls (n=72) 10-0 12 24 36 48 Overall survival (months) Natural human plasmacytoid dendritic cells induce antigen-specific T-cell responses in melanoma patients. Cancer Res. 2013 Feb 1 2013;73(3)

Conclusions Human pdc can be isolated and stimulated according to GMP FSME is a good substitute for CpG-C to trigger pdc via TLR, IFN-α, migration in vitro and in vivo, Ag presentation Commonly used vaccines are safe, good and cheap substitutes for synthetic GMP TLR ligands Clinical trials with peptide-loaded pdc are feasible (natural circulating DCs) No severe side effects nor toxicity has been observed pdc vaccine increase OS in stage IV melanoma patients

NATURE OUTLOOK. CANCER IMMUNOTHERAPY. Nature 2013, 504: 7480, S1-S16 OUTLOOK: SEARCHING FOR SYNERGY 1. Potentiate Ag-specific T cells 2. Block checkpoints inhibitors 3. Block immunosuppressive tumors environment

Therapeutic neoantigen-based vaccination Opportunities for immunotherapy in microsatellite instable colorectal cancer. Cancer Immunol Immunother. 2016 Apr 8. Westdorp H, et al.

Therapeutic approach for Lynch syndrome Opportunities for immunotherapy in microsatellite instable colorectal cancer. Cancer Immunol Immunother. 2016 Apr 8. Westdorp H, et al.

Clinical trials ongoing (based on DC) Hospital Clinic de Barcelona Protocol PEI NCT Eudra-CT Crohn intralesional 08-049 NCT02622763 2014-001083-35 MS * /NMO * 14-089 NCT02283671 2013-005165-39 CCR * 09-133 NCT01413295 DIPG * -DC 15-215 NCT02840123 2015-003362-84 CCR * + Avelumab 2016-003838-24 MS/NMO: multiple sclerosis; neuromielitis optica, CCR: colorectal cancer, DIPG: difuse pontine glioma DCs Phase II randomized trial of autologous tumour lysate dendritic cell (ADC) plus best supportive care (BSC) compared with BSC, in pre-treated advanced CRC patients colon. Eur J Cancer. 2016 Sep;64:167-74.

Take home message Cancer immunotherapies can eradicate tumours leading to complete and durable responses

Acknowledgements Hospital Clínic de Barcelona Department of Immunology NCMSL. Radboud University Department of Tumor Immunology TSF/BST (Transplant Service Foundation) Dept. Dermatology Radiology Surgery Microbiology Medical oncology Hematology

DCs Phase II randomized trial of autologous tumour lysate dendritic cell (ADC) plus best supportive care (BSC) compared with BSC, in pre-treated advanced CRC patients colon

61 patients assessed for eligibility 9 patients excluded 8 did not meet the inclusion criteria 6 positive virus (VIH, HBV) 2 no biopsiable disease 1 declined to participate 52 patients randomized ITT population 28 patients allocated to ADC+BSC 24 patients allocated to BSC 1 patient did not initiate treatment 27 patients received treatment 8 patients received potential active treatment+bsc 16 patients received BSC only

No clinical benefit PFS Progression-free survival (%) 100 75 50 25 BSC ADC + BSC 0 0 100 200 300 Days after randomization OS Overall Survival (%) 100 75 50 25 BSC ADC + BSC 0 0 200 400 600 800 1000 Days after randomization

DC expands/induce tumor specific T-cells 15000 p=0,0067 20 CPM 10000 5000 p<0,0001 % cells Th1 (Ifn-g +) 15 10 5 p<0,0001 0 0 PRE POST 40 POST 120 PRE POST 40 POST 120

OS is increased in patients with Ag-specific T cells Overall Survival (%) 100 80 60 40 20 OS p<0,02 ATMLR increase Non-ATMLR increase 0 0 200 400 600 800 1000 Days after randomization

Exogenous Ag presentation The poor ability to induce specific CD4+ T cell responses against exogenous antigens has been correlated with the lack of protein uptake by pdcs AIM To study the capacity of human pdcs to take up and present exogenous antigen

Vaccination protocol: monocyte-derived DC IL-4 GM-CSF KLH immature DC Maturation peptides monocytes mature DC - Metastatic melanoma - HLA-A2.1 + - gp100 + tyrosinase + KLH specific T cells (PBMC) KLH specific antibodies (post-vacc serum)

pdcs are able to present KLH. Specific T cell proliferation Humoral response to KLH No humoral response to KLH KLH-Alexa KLH-Alexa 3 H incorporation (cpm * 10 4 ) 3 H incorporation (cpm * 10 4 ) 16 14 12 10 8 6 4 2 0 16 14 12 10 8 6 4 2 0 * ** 1/20 1/100 pdc/pbl ratio 1/20 1/100 control KLH J Exp Med. 2006 Jul 10;203(7):1629

Endocytic receptors on freshly isolated pdcs CD32a DEC 205 CD36 BDCA-2 MHC class I 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 DC-SIGN MR DCIR Dectin-1 Isotype DEC-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells. Eur J. Immunol 2011 Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-α production. Blood. 2008

pdcs take up exogenous antigen Magnetic isolation BDCA-4 beads Purity (>95%) BDCA-2 pdcs from blood of: Normal donor Non-vaccinated 1.5 % Mono-DC (KLH) Vaccinated patients 36 % CD123 Specific antibodies against KLH 100 101 102 103 104 10 0 10 1 10 2 10 3 10 4 KLH-Alexa 488 10 0 10 1 10 2 10 3 10 4 NO NO YES

pdcs are able to take up KLH (immune complexes post-vaccination serum dependent) pdcs from normal donors + Serum from same patient (before/after vaccination) 1 % serum 5 % serum 20 % serum 23 % 55 % 58 % Before vaccination After vaccination KLH-Alexa 488 Are FcR involved?

KLH uptake is mediated by FcγRII/CD32 CD32a expression KLH-Alexa 48 % Irrelevant protein 5 % 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 Isotype + KLH-Alexa Anti-CD32 + KLH-Alexa 45 % 20 % 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 Are pdcs able to process and present KLH to specific T cells?

Blockade of PD-1 or CTLA-4 Signaling in Tumor Immunotherapy Antoni Ribas. Tumor Immunotherapy Directed at PD-1. N Engl J Med, 2012 366;26

Checkpoints inhibitors and new therapeutic opportunities CTLA-4 PD-1 New therapeutic approaches Anti-CTLA-4 Hard wired Targets CD28 pathway Expands clonal diversity Can move T cells into tumor Disease recurrence after response is rare Anti-PD-1 Induce resistance Targets TCR pathway Does not expand clonal diversity Does not move T cells into tumors Disease recurrence after response is significant