Virus

Cross-presentation

Tolerance
X X
Exogenous pathway
CD8 In draining LN

Immunity Innate
activator-”danger
” signals
DC
CD4
 Tumor Immunology
• Does it exist?
i.e., does the immune system recognize and
eradicate cancer cells? Is there any evidence for
immunological surveillance (Burnett and Thomas)?
• How can the immune system recognize cancer if it
is essentially self-tissue? (Tolerance)
• If it does not- can it be made to do so?
(Immunization designed to Break Tolerance)
Where is the danger-the innate activator?
 The Good News/Bad News Story
The immune system can destroy self-tissue quite
effectively in autoimmunity, and in a tissue-specific
(antigen-specific) manner: (thyroiditis, hepatitis,
pancreatitis (diabetes), vitiligo, ITP, AIHA, gradt rejection
etc.). So, self-tissue destruction can be potent.
• Are there ongoing anti-tumor immune responses in
patients with cancer?
– Spontaneous remissions are rare but can occur, renal cell CA,
melanoma, and are associated with anti-tumor Abs and CTLs.
TIL cells (tumor infiltrating cells) include CTLs that
recognize melanoma antigens/peptides (6/11
patients). But these CTLs were anergic:could not
kill targets or produce -IFN. Many patients make
anti-tumor antibodies, but are mostly IgM-will not
efficiently induce effector responses-and may
indicate a lack of T cell priming.
•  So..the good news is that immune
recognition of tumor antigens occurs but the
bad news is that this occurs without
activation of immune effector responses.
 More good news/
Evidence for Immunological Surveillance
Humans
• Increased incidence of malignancies in HIV patients: EBV
lymphoma, KS, squamous cell CA –but many of these are virally
induced malignancies; this merely shows that eliminating a T
cell response against viral antigens allows for the outgrowth of
virally-transformed cells. Common variety neoplasms (colon,
breast, prostate, lung, etc.,) are not increased.
•  In transplant associated EBV lymphomas (presumably arise
after the loss of EBV specific CTLs associated with T-cell
depleted allo-BMT. Cures are achievable by infusion of donor T
cells (reconstitute CTL response). Again loss of an anti-viral
responses is implicated. (post-transplant patients are also at
increased risk for melanoma and sarcoma).
Immunosurveillance: Tumors which Evolve in Lymphocyte Deficient
Hosts are Rejected in WT Mice
100%
RAG-/-
Tumor Incidence

Tumor (Sarcoma) Incidence is

WT Increased in MCA-treated
Lymphocyte Deficient Mice
0%

Tumor:
WT origin Tumors which
Tumor Size

developed in
RAG-/- hosts are
RAG-/- REJECTED in
origin WT Recipients

Host: RAG-/- WT
Immune Surveillance: Tumor Cell Expression of IFN Receptor is
Required for Lymphocyte-Mediated Tumor Rejection

100%

Tumor Incidence IFNR-/-

after MCA
Treatment

WT
0%

-------------------Transplanted tumor-------------------------------------
IFNR -/- IFNR -/-
WT IFNR-/- transfected transfected
with IFNR with IFNR
Tumor Size

Host: WT WT WT RAG-/-
Immune surveillance:
1. Innate system
NK, NKT,
gamma/delta T cells

IFN-
IL-12 (APC)

2. Functional
conventional T cells
 More good news/
Evidence for Immunological Surveillance
• In mice, absence of IFN-R, STAT1, IL-12, perforin,
RAG, NK cells: All of these genetic deficiencies have an
increased incidence of MCA (carcinogen) induced
malignancies.
Evidence that IFN-induced antigen presentation by
tumor cells provides immunity (as with viral immunity).
IFN-R -/- tumors grow in WT mice, unless transfected
with TAP. Highly immunogenic tumors emerge in RAG
-/- mice; these tumors grow in RAG -/- (in absence of
immune selective pressure) but are rejected in WT mice
(in presence of normal immune response).
Macrophages are primary source of IL-12 which induce
NK and T cell production of IFN-. (activates STAT1)
 Model of Innate Recognition and Initiation
of the Adaptive Antitumor Immune Response

Amplification of innate and link to

“danger”= invasion (inflam. adaptive response
response)
+ “stress” ligands of NKG2D

Apoptosis provides antigen

Elimination by
delivery to DCs
adaptive response
Immunization with Tumor Cells Can
Induce Protective Immune Response
Tumor Antigens Are Unique to Individual Tumors
Immunized Tumor
A B C D E F G H I
A
B
C
Tumor
D
Challenge
E
Protection F
G
No
protection H
I
 Candidate Tumor Antigens
Antigen Class Antigen Advantages/
Disadvantages
Whole Cell Protein lysate or Universal
tumor RNA based (Autoimmunity may be a problem)
expression
“Customized” therapy are required for
Peptide, DNA or these approaches. For whole proteins
Antigen- recombinant “antigen profile” of each tumor is
required. Peptides require additional
Specific protein
info. of indiv. HLA-type.
Antigenic modulation or loss (overcome
by attacking multiple targets and
antigens required for transformed
phenotype).
 Candidate Tumor Antigens..many
more to come through genomics
• Shared Tumor Antigens (common across tumors and
tumor types) Allows single therapy to be applicable for
many patients
1. Cancer/testes genes
2. Differentiation associated antigens
3. Others including gangliosides, MUC-1, etc.,
• Unique Tumor Antigens (requires tumor specific therapy)
Antigenic modulation would potentially interfere with
malignant phenotype.
1. Overexpressed proto-oncogenes: EGFR, HER2
2. Point mutations: ras, -catenin, CDC27, CDK4, Bcr/Abl
3. Viral Antigens: Human papilloma virus, EBV
 Antigen Class Antigen Malignancy
Immunoglobulin Idiotype B lymphoma, MM
TCR T cell lymphoma
Mutant ras Colorectal, lung, bladder,
Tumor Specific Antigen Head and neck cancer
Mutant p53 Pancreatic, Colon, Lung
p21-/bcr-abl fusion CML, ALL

Developmental Antigens MAGE-1, MAGE-3, GAGE family, Melanoma but also in

20 genes on the X chromosome colorectal, lung, gastric
(cancer/testes genes) Telomerase Various
Human Papilloma Virus Cervical, penile cancer
EBV Burkitt’s lymphoma,
Viral Antigens nasopharyngeal Ca, post-
Tx lymphoproliferative
Tissue-specific self- Tyrosinase, gp100,trp-1, trp-2 Melanoma
antigens Prostatic acid phosphatase, PSA Prostate
Thyroglobulin Thyroid
(Differentiation antigens)
-Fetoprotein Liver Cancer
Over-expressed self- Her-2/neu Breast and lung cancer
antigens CEA Colorectal, lung, breast
Muc-1 Colorectal, pancreatic,
ovarian, lung
 IMMUNE RECOGNITION

Tumor Evasion
Tumor cells are poorly immunogeneic

Therefore cross-priming required

(overcomes obstacles 1-4)

Poor APCs
1) Often no class I
2) No class II
3) No costimulatory molecules
4) Few adhesion molecules
5) Antigenically largely self
 IMMUNE RECOGNITION
Cross-Priming
• Host somatic cellular antigens (i.e.not soluble antigens)are
able to be presented to immune system by host APCs.
• True for viral antigens and cancer antigens.

Phagocytosis

Antigenic processing and

Dendritic presentation of antigen
Cell on class I and II

Immature DC Activation ?? Mature DC

DC Maturation
 Maturation Factors
• T cell signals (encounter with specific
Memory CD4 cell): CD40L
• Microbial stimuli: TLR ligands: LPS,
hypomethylated DNA (CpG), dsRNA (poly
dI:dC), peptidoglycans, StAg,
• Inflammatory Cytokines: TNF, IFN,
(products of either M, NK or T cells)
Effective antigen presentation by “cross-priming”
enhanced by DC activation/maturation (CD40L,
TNF, others)
• Peripheral immature DCs migrate to LN upon activation by
antigen/cytokines where they may encounter T cells.
• Maturation marked by transition of highly
phagocytic/endocytic cell to a poorly phagocytic/endocytic
cell.
• Upregulation of antigen processing and surface expression of
class I and II molecules
• Upregulation of cytokines, chemokines, co-stimulatory
molecules CD40, B7 (CD80,86) and adhesion molecules
(ICAM-1) for interaction and activation of antigen-specific T
cells.
 IMMUNE RECOGNITION
Cross-Priming: Induction of
Anti-tumor T cell response
Provide TH1 or 2
IL-2 Help for B cell
CD28
CTL
CD8 Ab Responses
TCR
CD4
TCR CD4 TH1

ClassI Class II CD40L

B7 +peptide
+
peptide CD40
APC Ag Processing/ Endocytosis/ Tumor Cell
Dendritic Cell presentation phagocytosis
of peptides
 Effector Mechanisms
CD8 CTL Can Recognize
Class I –peptide Complex and
Induce Tumor Lysis and Apoptosis

Granule exocytosis: CD8

Perforin/granzyme CTL
TCR

asL
Class I - F
s
+ peptide Fa

Tumor Cell
 Effector Mechanisms
NK Cells Can Recognize
Class I Negative Cells
and Induce Tumor Lysis
and Apoptosis
Granule exocytosis: KIR NK
Perforin/granzyme

Class I
X - F asL
s
Fa
Yet, class I loss is common
Tumor Cell in cancer.
Lack of activation of NK via
activating NK receptors?
Cytokine “milieu”?
 Effector Mechanisms
Macrophages are Cell-Mediated Effectors

TNF (+ other TNF-family members)

NO, O2•, proteases CD4
TCR CD4 TH1

CD40L

Class II
+ peptide Cytokine-
CD40 Mediated
Activation
Macrophage IFN-
GM-CSF
TNF
 Effector Mechanisms
Antibody Bound Targets Induce Myeloid Cell Tumor Cyto-
toxicity Through Fc Receptors +/or Complement Receptors
Y

Y
Tumor

Y
ADCC, phagocytosis, Cell
release of inflammatory mediators C3b

Y
(NO, O2•, proteases, TNF, etc.,)

Macrophage
 Effector Mechanisms
FcR Mediated NK Cell –ADCC
Y

Y
Tumor

Y
Y
Cell

Y
ADCC

NK Cell
 Tumor Evasion: Two separate
problems
• Tumor antigens are not recognized by
immune response-poorly immunogenic
(Immunologically ignorant).
• Tumors are resistant to or inhibit immune
cytotoxic responses.
(active suppression—either dampen
“priming” or avoid/inhibit/resist effector
cell function).
 Bad News/Tumor Evasion
Resistance to Effector Response
• Access to tumors may be limited by poor vascularity.
• Intrinsic resistance (anti-apoptotic genes).
Resistance to death receptor pathways: Reduction of Fas receptor
or enhanced expression of c-FLIP by tumors may render
tumors resistance to fas-mediated apoptosis. Similarly, tumors
commonly lose TRAIL receptors or express “decoy” receptors.
Upregulaton of “survival” pathways…akt, Bcl-2.
• Tumor cell or Tumor-associated-macrophage production of
local factors (TGF-, IL-10) that suppress T cell responses and
DCs (VEGF, and TGF, IL-10)
 More Bad News/Tumor Evasion
Resistance to Effector Response
• 2 pages of problems…not good
• FasL expression on tumor cells may induce cell death of Fas + T
cells.
• Conventional T cells may be suppressed by Treg cells or by CTLA4
(early clinical promise with CTLA4Ig).
• Antigen modulation (antibody-mediated endocytosis of surface
antigen)
• Loss of tumor antigen expression: Tumor heterogeneity (need to
target multiple antigens)-and possibly proteins essential for
transformation/growth.
• Loss of antigen presentation capacity by tumor
 Alterations in
Antigen Processing
(Loss of function
analogous to
tumor suppressor loss TCR
-tumor progression?)
CTL

Proteosome, TAP loss,

X Class I loss/ reg’n
Frequency
31-70%
2M loss, Class I loss
or  upregulation
TAP/Proteosome(LMP2,7)10-80%

Tumor Cell IFN-gammaR signaling defect

(rare)
*associated with metastatic and
 Immunological Intervention:
Early Successes
• Cooley’s toxin (gram + bacteria injected into tumor
sites): local inflammatory rxn and systemic toxicity
(fever, sepsis syndrome) associated with occasional
tumor remissions (bacterial product induced production
of IL-12, IFN-, TNF –enhanced antigen
presentation??)
• Systemic cytokines (IL-2, IL-12, IFN-) 1980-90’s.
Occasional responses (shrinkage in 5-15% of cases) with
high toxicities. Higher responses for IFN- in CML and
hairy cell leukemia; CML remissions associated with
anti-PR1 (proteinase in CML cells) T cell responses.
Strategies for induction of anti-tumor Immune Responses
-Passive-
• Adoptive transfer of T cells: Antigenic specific T cell clones-requires
HLA-restricted “customized” therapy or cytokine-enhanced antigen-non-
specific T cells (LAK cells). Has worked for EBV lymphoproliferative
disorders.

• Monoclonal and engineered antibodies:

1. Humanized/chimeric mAbs: Herceptin (anti-HER2), Rituxan (anti-CD20), anti-
idiotype (custom therapy), anti-EGFR (Erbitux), CAMPATH (anti-CD52), anti-VEGF
(targets neovasculature, Avastin).
2. Immune conjugates (“smart bombs”) mAb-toxin (Mylotarg: anti-
CD33 calicheamicin), mAb-chemo, mAb-isotope (anti-CD20 Zevalin and
Bexxar).
Potential Cytotoxic Mechanisms
of Anti-Tumor Antibodies
 Monoclonal Antibody
Therapeutics in Cancer
Rituxan (anti-CD20) Herceptin (anti-HER2)
• High response rate in B
cell lymphoma (>70%).
• Lower response rate in
breast cancer (15%).
• Synergy with • Synergy with chemo (60%)
chemotherapy or XRT. or XRT.
• Recognizes B cell marker • Recognizes EGF-like
regulating B cell receptor regulating cellular
activation. proliferation (ERBB2).
• Induces growth • Induces growth
arrest/apoptosis in vitro. arrest/apoptosis in vitro.
Monoclonal Anti-tumor Antibody Approaches in Cancer
 Strategies for induction of anti-
tumor Immune Responses
ACTIVE IMMUNIZATION

Goal is to define tumor antigens and then use them in an

immunostimulatory fashion.
How to induce immune response and break tolerance:
Essentially “the dirty little secret” of immunologists-the
adjuvant effect;effective immunization usually requires
mixing antigen with agents which promote uptake of antigen
by APCs as well as activate and recruit APCs to vaccine site
(e.g. Alum or Complete Freund’s Adjuvant: mineral oil/water
emulsion + heat killed bacillus).
 How to present antigen: clinical trials
• Systemic cytokines (e.g.IFN); upregulate HLA/antigen processing, mature
and activate APC
• Whole cell and adjuvant
• Tumor antigen protein or peptide and adjuvant
• Peptide and cytokines
• Turn cancer cell into an APC or a recruiter of APCs: transfect/infect tumor
with costim. gene (B7) or with cytokine gene (GM-CSF), DC tumor cell
fusion.
• Gene gun (DNA vaccination:tumor specific gene+/-costimulatory+/-cytokine
genes)
• Autologous DC’s “pulsed” with protein, peptides etc. Attempts to deliver
tumor peptide for cytosolic class I loading in activated DCs .
 Manipulation of DCs for Immunotherapy
• Autologous DC’s “loaded” with
Peptides of tumor antigens; (early 10-30% partial response rate in
advanced prostate CA and melanoma) practical problems:lack of
knowledge of 1) tumor antigens 2) HLA-restricted (available only for the
most common HLA-types, 3) antigenic modulation most likely results in
evasion for a small # of epitopes)
Known recombinant tumor antigens (whole protein) (Idiotype for B
cell lymphoma works but laborious)
Antigen non-specific approaches: Tumor lysates, Apoptotic bodies, RNA
encoding known tumor antigens, RNA derived using subtraction libraries,
DNA encoding known tumor antigens, Tumor-DC fusions
• DC delivery into tumors
Mobilization using Flt-3