Immunity and Cancer. Doriana Fruci. Lab di Immuno-Oncologia

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1 Immunity and Cancer Doriana Fruci Lab di Immuno-Oncologia

2 Immune System is a network of cells, tissues and organs that work together to defend the body against attacks of foreign invaders (pathogens, cancer cells, transplant tissues and often self tissues as well).

3 Most of precancerous cells die before they can cause cancer cells carrying mutations that destroy themself cells carrying mutations are recognise as abnormal by the immune system and eliminated Cancer is a disease which manages to avoid the immune system defence

4 Evidence for the role of immune system in tumor rejection 1. Increased incidence of i) virally-induced tumours in immunosuppressed patients, ii) lung carcinoma in cardiac transplant patients, or iii) de novo malignant melanoma in organs transplant patients 2. Higher incidence of cancer after immunodeficiency (AIDS) 3. Spontaneous regression 4. Reverse correlations between tumor infiltrating lymphocytes and overall survival

5 The Cancer-Immune Cycle Priming and activation 3 4 Trafficking of T cells to tumors Cancer antigen presentation 2 Lymph Node Blood vessel Tumor microenviroment 5 Infiltration of T cells into tumors 1 Release of cancer cell antigens Cancer testis antigens Differentiation antigens Neoantigens 7 Killing of cancer cells 6 Recognition of cancer cells by T cells Antigen expression Chen and Mellman, Immunity 2013; Nature 2017

6 Tumor microenvironment

7 Tumors can evade immune destruction Inhibiting T cell activation by dendritic cells Lymph Node Blood vessel Tumor microenviroment Blocking T cell infiltration into tumor Disrupting antigen detection Suppressing cytotoxic T-cell activity Antigen expression MHC-I TAP-1 B2m

8 PD-L1 inhibits cytotoxic T-cell activity in the tumor microenviroment

9 The cellular basis of tumour immunology CTL - tumor cell interactions

10 The cellular basis of tumour immunology NK - tumor cell interactions

11 The cellular basis of tumour immunology NK - tumor cell interactions

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13 MHC class I structure

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15 KIRs are sensitive to the peptides bound to MHC class I α 1 helix peptide α 2 helix TCR binding site KIR binding site Adapted from Parham et al. Phil. Trans. R. Soc. B 2012;367:

16 MHC class I Antigen Processing and Presentation

17 CD8 + T cell NK cell TCR KIR NKG2A pmhc Ia pmhc Ib cell membrane protein ERAPs ER Proteasome

18 Chr13C1 ERAAP ERAAP CAST Chr5q15 CAST LNPEP ERAP1 ERAP2 ERAP1 ERAP1-a 5 - poly(a)3 - N - GAMEN HEXXH(X)18E HDPEADATG -C

19 ERAP1 structure Domain I Domain II Substrate binding to regulatory site Domain III Domain IV Open conformation Less active Closed conformation Active Nguyen et al., 2011; Kochan et al., 2011; Stamogiannos et al., 2015

20 Molecular ruler of ERAP1 Chang E. et al. Proc Natl Acad Sci U S A. 2005

21 ERAP1 is naturally polymorphic ERAP1 disease association Cancer Cervical carcinoma Classical Hodgkin lymphoma Autoimmune disease Ankylosing spondylitis B*27 Psoriasis C*06:02 Behçet disease B*51 Birdshot chorioretinopathy A*29 Type 1 Diabetes Multiple sclerosis Juvenile Idiopathic Arthritis Stratikos E. et al. Front Oncol 2014 Hypertension

22 In the absence of ER peptide trimming, the repertoire of pmhc class I complexes is severely disrupted ERAAP-KO Blanchard and Shastri. Curr Opin of Immunol. 2008

23 Question Does interference of ERAAP affect tumor immunogenicity and tumor growth? ERAAP + ERAAP - Subcutaneous injection of cells Wild-type cells sieraap cells syngeneic mice Cancer Res, 2011, 71(5):

24 Silencing of ERAAP affects in vivo tumor growth of RMA lymphoma cells ERAAP+ ERAAP- RMA cells RMA sieraap cells sieraap un sc ERAAP ERp57 RMA RMA sieraap CD8+ Subcutaneous injection of 104 cultured cells C57BL/6 mice NK1.1+ Cifaldi et al., Cancer Res. 2011

25 Which is the lymphocyte population involved in this rejection?

26 Rejection of RMA-siERAAP cells requires NK1.1 +, CD4 + and CD8 + T cells Subcutaneous injection of 10 4 cultured cells C57BL/6 mice immunodepleted for CD4, CD8 and NK1.1 cells ip injection of mabs to CD4, CD8, NK1.1 days * * sc injection of cells Depletions were confirmed on splenocytes by flow cytometry 45 * Cifaldi et al., Cancer Res. 2011

27 NK cells are rapidly recruited in the peritoneal cavity of mice challenged with RMA sieraap cells Intraperitoneal injection of 106 cultured cells C57BL/6 mice immunodepleted for NK1.1 cells ip injection of mabs to NK1.1 days ip injection of cells Cifaldi et al., Cancer Res. 2011

28 Replacement of endogenous peptides with high affinity peptides restores the NK-protective effect of MHC-I + + ERAAP - ERAAP - RMA sieraap cells high affinity peptides LY49C NK cell - - Ly49I high affinity peptides Cifaldi et al., Cancer Res. 2011

29 In the absence of ERAAP Tumor cell

30 In conclusion (I) This work provides the first evidence that altered ER peptide trimming affects tumor growth by inducing T and NK cell-mediated immune responses Inhibition of ERAAP affects in vivo tumor growth of RMA cells Rejection of RMA-siERAAP requires intact NK cells, CD4 and CD8 T cell populations and depends on the MHC class I-peptide repertoire Replacement of endogenous peptides with high affinity peptides restores an NK-protective effect of MHC class I molecules through the Ly49C/I NK inhibitory receptors This study highlight the possibility of using ERAP1 as a novel biological target for cancer immunotherapy

31 ERAAP ERAP1 Does ERAP1 inhibition affect human anti-cancer immune responses? Exploiting ERAP1 modulation for cancer immunotherapy

32 Inhibition of ERAP1 activates NK-cell recognition in response to DAOY cells by impairing engagement of pmhc-i complexes with inhibitory receptors ERAP1 + Tumor cells Genetic inhibition Drug (Leu-SH) ERAP1 - + KIR NK cell - - NKG2A Lysis Cifaldi et al., Cancer Res. 2015

33 C*07:02 KIR2DL2/3 NK cell ERAP1 - DAOY cells KIR2DL1 NK cell B*57:01 C*06:02 KIR3DL1 NK cell What are the inhibitory receptors involved in NK cell activation in response to DAOY-shERAP1 cells? 1. Binding of KIR fusion proteins to DAOY cells 2. Aggregation of KIRs at the interface between NK and DAOY cells 3. CD107a expression in distinct NK-cell subsets

34 Inhibition of ERAP1 decreases KIR binding to DAOY cells and KIR aggregation at the interface between DAOY and NK cells Cifaldi et al., Cancer Res. 2015; 75:

35 C*07:02 KIR2DL2/3 NK cell ERAP1 - DAOY cells KIR2DL1 NK cell B*57:01 C*06:02 KIR3DL1 NK cell What are the inhibitory receptors involved in NK cell activation in response to DAOY-shERAP1 cells? 1. Binding of KIR fusion proteins to DAOY cells 2. Aggregation of KIRs at the interface between NK and DAOY cells 3. CD107a expression in distinct NK-cell subsets

36 Gating strategy used to evaluate the contribution of single inhibitory KIRs Cifaldi et al., Cancer Res. 2015; 75:

37 All inhibitory receptors are responsive to ERAP1 inhibition in DAOY cells Cifaldi et al., Cancer Res. 2015; 75:

38 What is the potential effectiveness of ERAP1 inhibition in clinical applications?

39 LCLs + Leu-SH Leu-SH-LCLs + NK cell In autologous or allogeneic setting ERAP1 inhibition affects NK cell recognition of allogeneic LCLs regardless of the involved KIR/ KIR ligand matching Strong killing No killing Cifaldi et al., Cancer Res. 2015

40 CD8 + T cell NK cell killing TCR MHC Ia KIR NKG2A MHC Ib killing cell membrane ERAP1 regulates innate and adaptive immunity by trimming peptides for presentation by MHC class I molecules ERAPs ER Proteasome

41 In conclusion (II) We provide the first evidence that genetic and pharmacological inhibition of ERAP1 in human cells perturb the engagement of inhibitory receptors by pmhc class I complexes, thus leading to NK cell killing. This mechanism depends on the peptide repertoire, because replacement of endogenous MHC class I-bound peptides with high affinity peptides restores an NK-protective effect. All ikirs tested are responsive to ERAP1 suppression in DAOY cells. ERAP1 inhibition significantly enhanced NK cell-mediated killing of LCLs in autologous and allogeneic settings regardless of the involved KIR/KIR ligand matching. These findings identify ERAP1 as a novel biological target in regulating human NK cells for cancer immunotherapy.