Anti-cancer T cell immunity

SMC6152 et BIM6028 EXMD604D

Mar 15, 2018

Woong-Kyung Suh, Ph.D.

IRCM woong-kyung.suh@ircm.qc.ca

Steve Jobs (1955-2011) Ralph Steinman (1943-2011)

What is common between the two?

Died of pancreatic cancer

Outline Part I • Cancer and immunity • Antigen presentation: virus vs. tumor • Specificity: structure of antigen-T cell receptor • Context: T cell costimulation • T cell receptor signaling pathways Part II • Immunoediting: 3Es • Cancer immune-evasion mechanisms • Anti-tumor immunotherapies: promises and challenges

Douglas Hanahan , Robert A. Weinberg (2000) The Hallmarks of Cancer Cell 100: 57-70

The hallmarks of cancer - 2000

Douglas Hanahan , Robert A. Weinberg (2011) Hallmarks of Cancer: The Next Generation Cell 144: 646-674

Hallmarks of cancer - 2011

Autoimmunity Allergy Immunodeficiency cancer

tolerance immunity

To react or not to react?

Specificity (foreign vs. self) Context (innocuous vs. dangerous)

T cell

Antigen presenting cell (APC: dendritic cell)

MHC restriction: T cells only recognize antigen on the surface of another cell, an APC

From Yewdell and Tscharke Nature 2002 418.923

MHC Major Histocompatibility complex proteins present peptides to T cells 2 kinds of MHC Proteins MHC I- CD8 T cells intracellular antigens MHC II- CD4 T cells extracellular antigens

Tania Watts, U of Toronro

Comparison of MHC I and II peptide loading pathways

David Williams U of Toronto

MHC class I assembly and presentation

• Watch a movie in YouTube. http://www.youtube.com/watch?v=VPvCekgPwRI

Class I Assembly in the Endoplasmic Reticulum

sec61 pore

Peptide Loading Complex

David Williams, U of Toronto

TAP associates with β2M associated Endo Hs form of class I

IP:

-“Pulse” EL4 cells with 35S-Met, 5 min -“Chase” with “cold” Met media for up to 4 hrs -Immunoprecipitate MHC class I molecules (Kb or Db) or TAP-class I complexes -Digest immunoprecipitates with endoglycosidase H -Resolve in SDS-PAGE -Autoradiogram

Addition of peptide causes dissociation of TAP/MHC I:

Add Kb binding Peptide- Kb dissociates from TAP complex and stable peptide loaded Kb form appears

Add Db binding Peptide, Db dissociates

Thermostable- detected by MHC I IP

Class I Assembly in the Endoplasmic Reticulum

sec61 pore

Peptide Loading Complex

David Williams, U of Toronto

Structure of human class I HLA antigen HLA-A2 Bjorkman et al. Nature. 329, 506, 1987

MHC I purified after cleavage from surface of human cell line with papain and purification (3-4mg from 200 liters of cells)

Note that although the α3 and β2M domains have an Ig superfamily fold, there is quite a different quaternary structure than in an Fab

Bjorkman Nature 1987

2 exons form single domain unidentified “peptide” Bjorkman PJ, Saper MA, Wiley DC. 1987. Structure of human class I histocompatibility antigen, HLA-A2. Nature 329:506–12 Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512–18

Co-purifying pepides- what are they?

H-2Db with 9-mer peptide bound (ASNENMETM) Young et al. (1994) Cell 76:39

X-ray Crystal Structures of MHC Class I Molecules

16

Interaction between the TCR and MHC class I molecule

Garboczi et al. (1996) Nature 384, 134.

αβTCR bound to HLA-A2 Variable loops of the TCR Vα and Vβ domains are centered over the peptide and α-helices of the class I H chain.

Peptide is buried in TCR-class I interface

Overall orientation of TCR is diagonal to class I groove. TCR fits between 2 high points on class I surface (top left and bottom right below). Ensures extensive contacts with both peptide and class I.

Footprint of TCR on MHC class I surface

Multiple contacts between residues of TCR and both peptide and class I α-helices. Of 46 contacts (H bonds and van der Waals contacts): - 18 are to class I conserved residues (confers general binding to class I)

- 9 are to polymorphic residues (confers allele specificity)

- 19 are to peptide

David Williams, University of Toronto

Context: costimulation

T cell APC

CD28

CTLA-4 B7.1/B7.2

TCR MHC

How to generate CTLs against virus that do not infect APCs? What about tumor cells?

David Williams U of Toronto

Presentation of Exogenous Antigens by MHC Class I Molecules: Cross-presentation

Pathway 1: Pfeifer et al. (1993) Nature 361:359. Song and Harding (1996) J. Immunol. 156:4182.

Why is this important? Priming naive CD8+ T cells against viruses involves costimulatory molecules that are absent on the majority of cells that are typically infected by viruses. The dominant cell that accomplishes such priming is the dendritic cell that acquires viral antigens largely by phagocytosis and then creates class I-viral peptide complexes at the cell surface. The same applies to tumor cells.

Process is: 1. Inhibited by cytochalasin D which blocks phagocytosis and by

chloroquine which inhibits endosome acidification and proteolysis (peptide production)

2. NOT inhibited by either brefeldin A (blocks ER to Golgi export) or

cycloheximide (protein synthesis inhibitor). Both block the conventional pathway.

3. NOT affected by loss of TAP transporter function Apparently, peptides produced in endocytic compartments either

bind to recycling class I in these same compartments or to "empty" cell surface class I.

X

David Williams, University of Toronto

Presentation of Exogenous Antigens by MHC Class I Molecules

Pathway 2: Kovacsovics-Bankowski and Rock (1995) Science 267:243.

Process is: 1. NOT inhibited by chloroquine 2. inhibited by loss of TAP function 3. inhibited by brefeldin A 4. inhibited by MG115 (inhibits peptide production by the proteasome) Thus, all indications are that the exogenous antigen must pass through the cytosol into

the conventional class I processing pathway.

David Williams, University of Toronto

Lipid Raft Lipid Raft

Fyn Lck

CD4

L A

T

PKC

MAPKKK

IκB NFκB

NFκB

PIP2

DAG IP3

[Ca2+]

Calcineurin

Calmodulin

NF-AT NF-AT

PLCγ1

IL-2

GADS SLP-76 VAV

GDP

GDP RHO

Actin reorganization SLAP130

Nck PAK

RAS

GRB2 RAF

MEK

ERK1/2

SOS

FOS JUN

Ras

ZAP 70

APC

T Cell

Csk

PAG

CD45

Dominik Filipp, IMG, Czech Republic

Summary – Part I • Dendritic cells process and present viral antigens or tumor

antigens to elicit antigen-specific T cell responses. • Endogenous antigens are presented by MHC class I

proteins to prime CD8 killer T cells whereas exogenous antigens are presented by MHC class II proteins to elicit CD4 helper T cell responses.

• However, exogenous antigens (viral particles, dead tumor cells) can be presented by dendritic cells through MHC class I proteins (cross-presentation).

• T cell receptor-peptide/MHC interaction allows discrimination between self and foreign antigens.

• Strength of costimulatory signals provide the context – dangerous or innocuous.

Break

Outline Part I • Cancer and immunity • Antigen presentation: virus vs. tumor • Specificity: structure of antigen-T cell receptor • Context: T cell costimulation • T cell receptor signaling pathways Part II • Immunoediting: 3Es • Cancer immune-evasion mechanisms • Anti-tumor immunotherapies: promises and challenges

Dunn et al. Nature Reviews Immunology 6,

836–848 (November 2006) |

doi:10.1038/nri1961

3Es in Immunoediting

Experimental test of “elimination” in mice _Create mouse models with altered immune cells or target cellls _KO _immune cells: RAG, _immune cell weapons: perforin, trail, IFN-γ _target cell responsiveness: STAT1, IFN-γR, proteasome subunits _mAb-mediated depletion: anti-Thy1.1, NK1.1, asialo-GM1 _activation of immune cells: α-GalCer, IL-12 _Score incidence, latency, and spectrum of _spontaneous tumors in aging mice _tumors induced by chemical carcinogens: MCA, DMBA, TPA _spontaneous tumors in p53 ko mice

Dunn et al. Nature Reviews Immunology

6, 836–848 (November 2006) |

doi:10.1038/nri1961

Evidence for tumor cells in equilibrium (dormant tumors)

Low dose MCA >eliminate mice (20%) that develop progressively growing sarcomas at d200 treat the mice having no tumors or stable tumor masses with antibodies that block components of innate or adaptive immunity measure tumor sizes Koebel C.M. et al. (2007) Nature 450: 903-907

Evidence for tumor cells in equilibrium (dormant tumors)

tumors outgrew if T cells or IFN-γ pathways were blocked but unaffected when innate immunity was blocked Thus, adaptive immunity prevents late MCA-induced sarcoma outgrowth Koebel C.M. et al. (2007) Nature 450: 903-907

Evidence for tumor immune escape

• Many established murine cancer cells induce anti-tumor immune responses but grow well when injected in immune competent hosts

• These escapes often down regulate MHC class I or secrete immunosuppressive factors

Questions

1) Do cancer cells provoke T cell attack? If so, how? 2) How do cancer cells avoid or subvert T cell attack? 3) How can we harness T cells to fight cancer?

Do cancer cells provoke T cell attack? If so, how?

• Carcinogens, or UV induces mutations. Some

of the mutations can give rise to tumor-specific antigens

• Rapid proliferation in the absence of tumor suppressors lead to DNA damage and mutations. Many mutations can be immunogenic .

Tumor specific T cell antigen (Rejection antigen)

Questions: 1) New tumor specific rejection antigens arise in nascent tumor cells? 2) Rejection antigens are sufficient to induce protective anti-tumor immunity? Approaches: Take tumor cells grown in immunodeficient (RAG2 KO) mice 1) Clone single cells, analyze their growth patterns in WT mice (progressor vs regressor), and identify “neoantigens” uniquely expressed by regressors 2) Express identified rejection antigens in progressor clones and see if this can convert progressors into regressors and induce anti-tumor T cells Mastushita et al. (2012) Nature 482:400-404

Progressor (20%)

Regressor (80%)

Single cell cloning

Mastushita et al. (2012) Nature 482:400-404

Are the escapers pre-existing?

Tumor specific T cell antigen (Rejection antigen)

-Identify tumor-specific mutations by exome sequencing -Choose potential class I binding epitopes -Predict affinity values -Choose mutant epitopes uniquely expressed in regressors R913L mutation in spectrin β2 is one of them (750 fold increase in binding affinity)

Mastushita et al. (2012) Nature 482:400-404

Tumor specific T cell antigen (Rejection antigen)

Estimation of tumor specific T cell antigens in human cancers

Neil, H. et al. (2008) Cancer Res. 68:889-892

• Analyzed 1,152 peptides with missense mutations in breast and colorectal cancer using epitope prediction algorithms

• Identified average ~10 novel and ~7 unique epitopes that can bind to human MHC class I molecule HLA-A0201

• Suggest that tumor cell destruction in situ (e.g. by chemotherapy) in combination with adjuvants may induce polyvalent anti-tumor T cell immunity

How do cancer cells avoid or subvert T cell attack?

• Decrease surface MHC class I molecules • Become insensitive to IFN-γ: no upregulation of

MHC molecules, TAP, and proteasome subunits. • Suppress anti-tumor immune cells: TGF-β, VEGF,

Indoleamine 2,3-dioxygenase (IDO) > recruit pro-tumor immune cells such as tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg)

How can we harness T cells to fight cancer?

• Engineer tumor-specific T cells in vitro and infuse back into patients: chimeric antigen receptor (CAR)

• Elicit endogenous T cell responses by boosting antigen presentation: ex vivo or in vivo dendritic cell therapies

• T cell immune checkpoint blockade

Stanley R. Riddell , Michael C. Jensen , Carl H. June (2012) Biology of Blood and Marrow Transplantation Volume 19, Issue 1, Supplement 2013 S2 - S5

Chimeric antigen receptor (CAR)

Extracelular: Single chain Fv to a tumor antigen Intracellular: Combination of TCR and costimulatory signaling motifs

CD19 CAR: a success story

Porter, D.L. et al. (2011) N Engl J Med 365:725-33

-CD19 is highly expressed in chronic lymphocytic B cell leukemia as well as normal B cells. -T cells were taken from CLL patients and modified by lentiviral transduction to express anti-CD19 CAR. -Reinfusion of modified T cells led to complete remission in one out of three patients.

DC-mediated cancer immunotherapies

Palucka, K and Banchereau, J (2012) Nature Reviews Cancer 12: 265-277

Chemotherapy inducing immunogenic cell death (ICD)

Kroemer, G. et al. (2012) Annu. Rev. Immunol. 31: 51-72

Immunogenic cell death induces anti-tumor T cell response

Kroemer, G. et al. (2012) Annu. Rev. Immunol. 31: 51-72

Checkpoint blockade: CTLA-4 and PD-1/PD-L1 pathways

• CTLA-4 or PD-1/PD-L1 blockade enhances anti-tumor T cell immunity

• Ipilimumab (anti-CTLA-4): first FDA approved immunotherapeutic drug (2011), humanized mAb that blocks CTLA-4. 15-25% of melanoma patients show complete remission. Being tested for lung cancer, prostate cancer, and others.

• Combination of CTLA-4 and PD-1 (or PD-L1) blockade provides synergistic effects.

• Combination of checkpoint blockade with irradiation or chemotherapies under clinical trial.

CTLA-4 & PD-1/PD-L1 blockade

Steve Jobs (1955-2011) Ralph Steinman (1943-2011)

Pancreatic cancer: How Nobelist Ralph Steinman beat the odds, but Steve Jobs didn’t By Janet Fang | October 10, 2011, 9:53 PM PDT

Jobs: Diagnosed pancreatic neuroendocrine tumor (curable by surgery) in 2003. But he insisted on alternative medicine 9 months before agreeing to get surgery. In 2009, cancer spread to liver and he had to get a liver transplant. To prevent graft rejection, he had to take immunosuppressant drug. Steinman: Diagnosed pancreatic adenocarcinoma (fatal) 1997. Chemotherapy combined with eight DC-based experimental immunotherapies. 4.5 yr survival with adenocarcimoma was extremely rare.

Summary

• Cancer cells do produce tumor-specific antigens and elicit anti-tumor T cell reaction.

• The immune system can function as an extrinsic tumor suppressor and influence the repertoire of outgrowing tumor cells (3E hypothesis).

• Immune system can be harnessed to fight cancer: dendritic cells and T cells are most promising.