10 31 2006 Oncology Cancer Immune Lecture

Roles of Tumor-Associated Antigens on Cancer Immunosurvillance and Tumor Escape

國家衛 生研究院所 　癌症研 究所 講員 :

施　能 　耀　博士

Key Events in Cancer Biology 1909-50s: Immune surveillance theory postulated by Paul Ehrlich and proven in animal models by Thomas & Burnet 1976:

Discovery of proto-oncogene derived from RNA tumor virus studies (Bishop & Varmus), i.e.c-Src

1970-80s: Discovery of tumor suppressor and “two-hit” hypothesis postulated by Knudson, i.e. Rb-1 gene 1991:

Cloning of human immunogenic tumor-associated gene, MAGE-1 from melanoma using tumor-specific CTL (Thierry Boon’s group)

1995-97: Cloning of human SEREX-defined tumor-associated genes using cancer patient’s serum (Sahin & Chen)

Progression of Tumor Formation

Initiation state

Initiate cell cycle progression

Promotion state

(1) Bypass cell cycle checkpoints

Immune surveillance

Tumor

(1) Immune tolerance (3) Insufficient immunity

(2) Loss of self-suicide ability (apoptosis)

Concept of Immune Surveillance in Cancer Biology: Evidence from Animal Study and Human Diseases

Immune Surveillance Theory Thomas and Burnet (1950s) proposed that our immune system evolved to recognize and destroy transformed cells before they grow into tumors. Mouse with MCAinduced tumor Tumor cells

No tumor growth

Isolated CD8+ T cells

Tumor growth

Eradication of tumor

Controversy on Immune Surveillance Theory Osias Stutman: 7/39 of immunocompetent CBA/H mice formed MCA-induced sarcoma with a mean time to tumor appearance of 95 days 5/27 of athymic nude mice formed sarcoma with a mean time to tumor appear of 90 days Rygaad and Povlsen: no differences in spontaneous tumor formation in 10,800 nude mice over 3-7 months Defects: 10. CBA/H strain mice are highly sensitive to MCA-induced tumor formation (distinct isoforms of aryl hydrocarbon hydroxylase in various inbred mouse strain) 2. Athymic nude mice are not completely immunocompromised (existence of functional NK and γδ T cells) 3. The monitoring periods of 3-7 months are too short to see tumor formation in functional intrinsic tumor suppressor systems

Key Findings Renew Interest in Cancer Immunosurveillance •

Endogenous IFN-γ protecting the host against the growth of transplanted tumors was shown in the following systems: --- lacking of IFN-γ expression in gene-targeting mice --- neutralizing mAb specific for IFN-γ --- lacking of STAT1 or IFN-γ receptor in gene-targeting mice

B. Perforin-/- mice lacking perforin expression was higher susceptible to MCA-induced tumor formation when compared to their wild-type counterparts. --- showed higher incidence of either MCA-induced sarcoma or spontaneous lymphoma

Successful Tumor Immunity in Early Stage of Human PND and in Clinical Prognosis Paraneoplastic Neurological Degeneration (PND): --- an autoimmune disease to cause nervous degeneration at a time when tumor remains invisible. Cause: autologous antibodies of PND patients recognize the same antigens that are expressed in the patient’s tumor and in normal neuron. Clinical prognosis: some patients with immune response against onconeural antignes have smaller tumors and longer survival than those with poor immune responses.

A Molecular Model for the Pathogenic Paraneoplastic Neurological Disease

* Adopted from K.Musunuru and R. B.Darnell. Annu. Rev. Neurosci. 24: 239 (2001)

1. What is the role of immune surveillance during cancer formation? 2. Can Immunotherapy be an alternative therapy for cancer patients?

The 3 “E” stages in Cancer Formation

* Adopted from Schreiber, R.D., etc Nature Immunol. 3:991 (2002)

A proposed model for the elimination phase of The cancer immuno-editing process

* Adopted from Schreiber, R.D. Nature Immunol. 3: 991 (2002)

Activation versus suppression during tumor progression

* Adopted from Khong and Restfio. Nature Immunol. 3:999 (2002)

Immune Effectors in Cancer Immunotherapy

Integrated immune response to human tumor antigens MHC class I

Tumor cell Gene

Cytotoxic T cell

Expression

CD8+ Processing

TCR

nti

ge n

Release of antigens

Presentation

tak eo fa

An ti

Antigenic peptides

gen

Help

Up

Antigen TCR

mIg Endocytosis

OR

Secreted antibodies (SEREX reagent) Dendritic cell

Endocytosis

CD4 CD4++ MHC class II Help

B lymphocyte cell

T helper cell

Specific and Non-specific Immunities in Cancer Immunosurveillance Specific immunity: • CD4+, CD8+ T lymphocytes • B lymphocytes • Professional antigen presentation cells (dendritic cells, etc.) Non-specific immunity: -- Nature Killing (NK), NKT cells, γδ T cells

Activation of cytotoxic activity of T cells to tumor cells

(perforin)

Apoptotic tumor cells release tumor-associated antigens Uptake by local antigen presenting cells (Macrophages or DCs) Antigen process and present to CD8+ T cells, which become activated Attack the living tumor cells by discharging perforin to lyze the cells

Stages of T cell Activation in vivo Antigen Process and Presentation

T cell activation

Activation of Effector

IL-2 / IFN-γ Perforin secretion

T cell recognition of a peptide-MHC complex

Two-signal hypothesis for T cell activation

* Adopted from Trapani, J.A. (2002). Cancer Cell: 169.

Molecular parameters for T cell activation Naïve T Adhesion molecules

Memory T

Activated status

Chemokines

GEMs

Threshold

Co-stimulatory molecules Un-phosphorylated PTKs ↑ level of cAMP ↑ Rap 1 activtion Ground status ↑ p27 kip1

Antibody-dependent Cell-mediated Cytotoxicity A. Cell-mediated phagocytosis: neutrophils and macrophages

FcγRI (CD64)

B. Cell-mediated cell lysis: NK cells (FcγRIII), Eosinophils (FcεRI)

FcγRIII

IFN-γ

(CD16)

Perforin

Antibody-dependent Complement-mediated Cytotoxicity

Binding of C1 to antigen-complexed antibody Spontaneous breakdown of C3, binding C3b to microbial surface

Formation of C3 convertase

Cleavage of C3 by cell-associated C3 convertase

Cleavage of C3 by C3 convertase

Binding of C3b to microbial surface and to form C5 Convertase (C3bBb)

Binding of C3b to Antigenic surface and To form C5 convertase (C4b2a3b)

Phagocytosis of opsonized Particles (C3b)

Activation of late steps of complement (C5 convertase)

Inflammation induced by (C3a, C4a, C5a)

Late steps of complememt activation and formation of membrane attack complex (MAC)

Roles of Tumor-associated Antigen on Tumor Escape

Tumor Escape Mechanisms A. Immunodeficiency of T cells (1) clonal deletion of tumor-specific T lymphocytes (2) defects in signaling pathways (3) T cell anergy or tolerance B. Immunosuppression induced by tumor cells (1) loss of MHC or co-stimulatory molecules (2) defects in antigen processes and presentation (3) secretion of apoptotic ligands or immune suppressive cytokines (FasL, TGF-β, IL-10., etc.) (4) Alteration in apoptotic signaling pathways

The Causes induce T cell Immunodeficiency

(1)Defects in TCR-mediated signaling pathways (2) Clonal deletion of tumor-specific T lymphocytes

Immune Surveillance Theory Thomas and Burnet (1950s) proposed that our immune system evolved to recognize and destroy transformed cells before they grow into tumors. Mouse with MCAinduced tumor Tumor cells

No tumor growth

Isolated CD8+ T cells

Tumor growth

Eradication of tumor

Formation of a specialized adhesion complex, immunological synapse, between T cell and antigen presenting cell

Activation of T cell requires multiple co-stimulatory molecules involved TH cell

Antigen presenting cell

Antigen peptide

CD2 LFA-1

LFA-3 ICAM-1

TCRCD3

MHC Class II

CD4 CD45R

CD22

CD28

B7

Molecular parameters for T cell activation Naïve T Adherent molecules

Memory T

Activated status

Chemokines

GEFs

Threshold

Co-stimulatory molecules Un-phosphorylated PTKs ↑ level of cAMP ↑ Rap 1 activtion Ground status ↑ p27 kip1

Activated TCR-mediated signaling pathways in T cells Acitivated TCR

Receptors

LCK/ZAP-70/FYN

PTKs Linker proteins Effectors 2nd messeges/ kinases Gene transcription Biological responses

LAT Ras

GADS PLC

MAPK Ca2 ＋ /PKC

SLP-76 Vav PKC-θ

Rac Cdc42

WASP Arp 2/3

Fos/Jun NFAT/NF-κB

IL-2 transcription

NcK

F-actin

Defects in TCR-mediated signaling in T cells obtained from Intracranial tumors

P56lck, but not p59fyn, protein levels are reduced in patient T cells

The tyrosine phosphorylation of PLCγ1 is reduced or undetectable in patient T cells

The intracellular [Ca2+] is greatly reduced in ionomycin-induced patient T cells

* Data obtained from Morford, L.A., etc. J. Immunol.159:4415 (1997)

B7-H1 Expression on p815 cells prevents rejection by syngenereic DBA/2 mice

* Adopted from Trapani, J.A. (2002). Cancer Cell: 169.

Mock/624mel

B7-H1/624mel

M15 CTL

Anti-PD-1

Anti-B7-H1 Mock/624mel

B7-H1/624mel+ mIgG1+ M15

Apoptotic cells (%)

Induction of M15 CTL Apoptosis by B7-H1 Expression in 624mel cells

Medium

Mock/624mel

B7-H1/624mel

Mock/624mel + M15 CTL

B7-H1/624mel+B7H1Ab+M15

＊ Adopted

data from Dong, H.-D., etc. Nature Med. 8: 793 (2002)

Expression of B7-H1 in Human Cancer Tissues Anti-B7-H1

Melanoma

Ovarian cancer

Lung cancer

H&E

* Adopted from Dong, H.-D., Chen, L.-P., etc. (2002) Nature Med. 8:793

T cell Immunosuppression induced by tumor cells

•

Secretion of apoptotic ligands or immune suppressive cytokines (FasL, TGF-β, IL-10., etc.)

•

Alteration in apoptotic signaling pathways

Fas-mediated Activation-induced T Cell Apoptosis Crosslinking of Fas by FasL

Binding of FADD to crosslinked Fas

Binding and autocatalytic Activation of caspase 8

Activation of effector caspases 3 / 6 / 7

cFLIP

Active caspase 8

Apoptosis cFLIP: cellular FLICE inhibitory protein

Putative Mechanisms for Inhibition of T cell-mediated Apoptosis following Ligation of Cancer Cells

or cFLIP

PI-3 kinase: phosphatidylinositol-3 kinase

MPR: Mannose-6-phosphate receptor PI-9: Protease inhibitor 9

* Adopted from Trapani, J.A. Cancer Cell:169 (2002)

Models for the Introduction of FasL-mediated T cell death after encounter with tumor cells

* Adopted from Khong and Restifo. Nature Immunol. 3: 999 (2002)

Inhibition of Cell Growth and Apoptosis in T cells by RCAS1 tumor antigen Expression of RCAS1 receptor in PBLs

200 U/ml IL2 20 U/ml IL2 0 U/ml IL2

GST control

RCAS1

GST

Induction of apoptosis in activated PBLs

Medium

GST-RCAS1

medium GST GST-RCAS1 Medium from 0 day culture

Medium from 2 days culture

Suppression of T cell Activation

Blocking the engagement between TCR and peptide-loaded MHC

Formation of a specialized adhesion complex, immunological synapse, between T cell and antigen presenting cell

Large surface molecules prevent T cell recognition of tumor cells

CMS5-CEA

CMS5-Neo

+ IFN-γ CTLs of DUC18 mouse DUC18 mice Tumor Growth

No tumor

Expression of CEA enhance tumorigenesis of CMS5 cells A.

Cytotoxicity assay 100

CMS5 CMS5-neo CMS5-CEA

75 % of Killing 50 25 0

1

2 3 4 5 Effector / Target ratio

6

B. In vivo growth of transfected CMS5 cells 120 High dose CMS5-CEA High dose CMS5-neo Low dose CMS5-CEA Low dose CMS5-neo

100 Tumor size (mm)

80 60 40 20 0

5

10 15 Days after injection

20

25

Conclusions from CEA Studies in CMS5 transfected cells

‧CEA with 7 Ig extracellular domains did protect CMS5 cells from killing by T cells of DUC18 mice, suggesting overexpressed CEA may enhance tumorigenesis.

‧ The killing of CMS5 tumor cells in vivo is mediated by specific immunity since both CEA-transfected or un-transfected cells grow well in SCID mice.