•Immunity and Tumors
•Cancer Immunotherapy
Immunity and Tumors
1890s
Coley treats patients with bacterial extracts
1950-1960s
Burnet and Thomas- Immune-surveillance Hypothesis
“Major function of the immune system is to recognize and
destroy arising malignantly transformed cells”
1957
Prehn and Main- The origin of modern tumor immunology
Pathogenesis of neoplasia
N
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o
n
r
i
m
t
a
i
DNA damage
l (chemical, phisical, biologic) a
t
i
o
n
differentiation proliferation
apoptosis
P
P
r
r
o
o
m Clonal evolution g
o Additional mutations r
DNA damage
e
t
s
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si
o
o
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n
proliferation
I tumori e la risposta immunitaria
I tumori derivano da tessuti normali
I tessuti normali non inducono risposta immunitaria
Esiste una risposta immunitaria contro i tumori?
L’immunosorveglianza
1959 Thomas e Burnet: l’immunosorveglianza
The immune system maintains vigil over both alien
microorganisms and altered somatic cells
Il tumore è antigenico ed immunogenico
1) Evidenze sperimentali:
Il trapianto di tumori in ospiti singenici viene rigettato, mentre il
trapianto di tessuti normali viene accettato
Il rigetto di tumori spontanei o indotti conferisce protezione
2) Evidenze cliniche:
L’insorgenza di tumori è piu’ alta in assenza di competenza
immunologica (immunodeficienze congenite o acquisite)
Evidenze di immunogeneticità dei tumori
Sarcoma
Vaccinazione con
cellule tumorali
Resezione
chirurgica
Topo naive
Nessuna crescita
Crescita del tumore
Topo vaccinato
Nessuna crescita
Conclusions from experiments on transplanted tumors
- The immune system of inbred mice can recognize antigens expressed
by tumor cells induced by chemical carcinogens
- Such recognition results in rejection of a subsequent challenge of the
same but not a different tumor in previously immunized animals
- Specificity and memory
- The immune cells but not antibodies can mediate this reaction
Evidenze sperimentali
dell’immunogenicità dei tumori
1. Presenza di cellule mononucleate nel siti di crescita del tumore
(linfociti T, natural killer, macrofagi)
2. Iperplasia dei linfonodi drenanti il sito di crescita del tumore
3. Evidenze di effetti dovuti a citochine pro-infiammatorie
direttamente sul tumore (indotta espressione di MHC II, ICAM-1)
4. Regressione spontanea di alcuni tumori.
Evidenze cliniche di immunosorveglianza
1) Inherited Immunodeficiency
2) Organ transplant recipients
3) Patients with auto-immune disorders
4) Second tumors in cancer patients
5) HIV infection
Evidenze cliniche di immunosorveglianza
1) Inherited Immunodeficiency
Syndrome
Immune defect
Tumors
X-linked
immunodeficiency
Impaired B cell
response to EBV
Non Hodgink’s
Limphoma
Wiskott-Aldrich
syndrome
Complex
multicompartment
defects
NHL, Acute myeloid
leukemia, Hodgink’s
disease
Common variable
immunodeficiency
Cellular and humoral
defects
NHL, stomach cancer
Experiments in gene-knockout mice lacking various components
of the immune system
- IFN-g deficient mice have a higher rate of both spontaneous and carcinogeninduced tumors
- Double IFN-g and Rag-2 deficient mice
- Perforin-deficient mice
- TRAIL-deficient mice
Role for NK cells
Tumor cell recognition by NK cells
Missing self recognition:
- Inhibitory receptors (KIR, CD94/NKG2A) bind directly to intact MHC class I
molecules
Recognition of induced self ligands as marker of abnormal self:
- Stimulatory receptors (NKG2D) bind to ligands expressed or up-regulated in
tumor cells and virally infected cells
- Ligands: MICA/B expressed on tumor cells of epithelial origin; Retinoic
acid early inducible protein (Rae1); H60
L’immunosorveglianza
presuppone l’esistenza di:
1. Antigeni tumore specifici/ tumore associati
2. Cellule effettrici in grado di riconoscere il tumore e
mediarne il rigetto
Conoscere l’identità degli antigeni tumorali è fondamentale per
sviluppare immuno-terapie antigene/tumore specifiche
Caratterizzare le cellule effettrici è fondamentale per poter
intervenire e manipolare la risposta immunitaria
Elementi critici nello sviluppo di
una risposta anti-tumorale
Risposta primaria
Risposta secondaria
Tumore
(sorgente di antigene)
Tumore
Cellule adibite alla
presentazione antigenica
Cellule adibite alla
presentazione antigenica
Linfociti T e B
Linfociti T e B
Colocalizzazione
Macrofagi, cellule NK, NKT
Anatomy of the adaptive
immune responses
Primary lymphoid organs:
bone marrow and thymus
Secondary lymphoid organs:
lymph nodes and spleen
Non lymphoid organs:
site of infection
Anatomy of the adaptive
anti-tumor immune responses
Primary lymphoid organs:
bone marrow and thymus
Secondary lymphoid organs:
lymph nodes and spleen
Non lymphoid organs:
site of tumor growth
Physiological condition
Tissue antigens
Tissue-specific antigens are ignored
Patological conditions
Lymph
Blood
Transforming event
Tumor antigens
or tumor cells
Blood
Tumor-specific T cells
Tumor-specific immune responses
The fine balance between
immune responsiveness and immune resistance
Spontaneous inflammation in
the tumor microenvironment
Immune stimulation
or inflammation
Tumor
regression
Tumor growth
Tumor
progression
Antigen-specific
immunization
Non specific
immune stimulation
Critical factors in adaptive immune responses
Proper selection of antigen specific progenitors
Secondary lymphoid organs
Appropriate timing
Proinflammatory stimuli
Shaping of the immune response over time and space
Main lymphocytes subsets
participating to anti-tumor responses
Th-1 CD4 T lymphocytes: helper cell, CD8, APC, killing
Th-2 CD4 T lymphocytes: helper cell, B cells
Tc-1 CD8 T lymphocytes: cytotoxic cell
B cells: Ab production
NK, NKT, gd T cells
Classification of tumor antigens
- Tumor-specific shared antigens/Cancer-testis antigens
Antigens encoded by genes expressed in variable proportion on
different cancers, but not in normal tissues except testis and placenta
- Differentiation tumor antigens
Antigens encoded by genes expressed in tumor cells and in normal tissue
- Unique tumor antigens
Antigens corresponding to peptides encoded by regions of ubiquitously
expressed proteins that are mutated in tumor cells
- Over-expressed tumor antigens
Antigens encoded by non-mutated genes that are expressed at different level
in neoplastic and normal tissue
- Viral antigens
Antigens derived from oncogenic viruses
Fong, L and Engleman, EG
Annu Rev Immunol, 18:217,
2000
CD4+ T cells are important for tumor rejection
- In vivo depletion experiments with antibody recognizing
different lymphocytes population
- Experiments using CD4-knockout mice
- Adoptive transfer of tumor-specific CD8+ and CD4+ T
lymphocytes
CD4+ T cells
in anti-tumor immune response
Lymphoid
organs
Peripheral
tissues
Priming phase
Tumor cell
Mature
dendritic cell
Tumor antigens
Draining
Lymph node
Immature
dendritic cell
MHC
Class I
MHC
Class II
Tumor
peptides
CD40 CD8+
CD40L T cell
Th2
Effector phase
Th1
CD4+
T cell
CD4+
CTL
B cell
Th1/Th2
Th1
Killing
Reactive oxigen
intermediates
Release of
granule contents
Macrophage
Tumor cell
Killing
CD8+
CTL
Effector mechanisms in cancer immunity
- Antibodies
- Coating with antigen, opsonization and phagocytosis by macrophages
- Crosspriming
- NK cells
-Lyse MHC mismatched cells, cells having low level of or lacking
MHC class I expression, cells expressing ligands of stimulatory receptors
- NKT cells
- Recognize glycolipid antigens by non-classical MHC molecules and
produce large amounts of type 1 or type 2 cytokines
- Macrophages and neutrophils
- Activated by tumor microenvironment, and CD4+ T cells
- Release of tumoricidal factors (TNF, nitrogen oxides), endocytosis of
malignant cells
- Cytokines
- T cells
Different mechanisms may be responsible for failure
to develop effective anti-tumor immunity in vivo
- Failure to develop immunity
Ignorance
-Tolerance induction
Anergy/Deletion
- Mechanisms of immune escape
Failure to develop efficient anti-tumor immunity
Tumor cell
Antigen uptake by
tolerance-inducing APC
T cell
APC
T cell
TCR
MHC-peptide
Tolerance
Receptor for
costimulatory
signals
Mechanisms of tumor immune escape
- Loss of MHC expression
- Down-regulation of antigen processing machinery
- Antigen loss variants
- Expression of local inhibitory molecules (FasL)
- Secretion of immunosuppressive cytokines
- IL-10, TGF-b
Cancer Immunotherapy
Strategies of antitumor immunotherapy
- Adoptive immunotherapy
- Monoclonal antibodies
- Active vaccination
-Vaccination against tumor
neovascularization
Strategies of antitumor immunotherapy
- Adoptive immunotherapy
- LAK
- TIL
- DLI
- CD8 clones
- Cell cloning technique
- TCR transfection
Adoptive transfer of IL-2 activated tumor
infiltrating lymphocytes (TILs)
Adoptive transfer of TILs expanded in vitro and high dose IL-2 following a nonmyeloablative conditioning regimen
Dudley ME et al. Science, 2002
Antigen specific T cells transfer
Adoptive transfer of antigen-specific CD8+ T cell clones
In vivo persistence, migration and antitumor effect of transferred
MART-1/Melan-A specific T cell
Yee, C. et al. PNAS 99: 16168, 2002
Strategies of antitumor immunotherapy
- Monoclonal antibodies
Monoclonal antibodies
Mechanisms of action:
- antibody-dependent cell-mediated cytotoxicity
- cross-presentation by immune complexes
Clinical studies
- anti-CD20 (B-cell lymphomas)
Monoclonal antibodies as magic bullets.
Strategies of antitumor immunotherapy
- Active vaccination
Goal of cancer vaccines
- To identify ways to break tolerance
- To identify resistance mechanisms
and ways to circumvent them
Vaccine design
- Targeting CTL responses
- Targeting CD4+ T cell responses
- Targeting multiple antigens and epitopes that cover a broad repertoire
of T cells
Undefined (cancer cell extracts, mRNA)
- Choice of the antigen
Defined
- Adjuvant
- Dose
- Route of injection
- Schedule
Different forms of cancer vaccines
- Cell based cancer vaccines
- Antigen specific cancer vaccines
- Dendritic cells vaccines
- Heat shock proteins vaccines
Cell-based cancer vaccines
Tumor cell as a source of antigen (autologous or allogenic)
Early generation:
- Killed tumor cells or tumor cell lysate mixed with adjuvants
such as BCG
Genetically modified tumor cells:
- Immunologically active genes
- MHC genes
- genes encoding membrane associated costimulatory molecules
(B7-1, B7-2)
- cytokines genes (IL-2, IL-4, GM-CSF)
Clinical trials:
- Several with limited success
Antigen-specific cancer vaccines
- Peptide vaccine
- Protein vaccine
- Recombinant viral vaccine
- Recombinant bacteria vaccine
- Nucleic acid vaccine
Peptide vaccine
Depends on loading of empty MHC class I molecules in vivo
Advantages
- Easy to manufacture in GMP conditions
Disadvantages
- May results in tolerance induction
Clinical trials:
- MAGE-3 presented by HLA-A1
Marchand M, et al. Int J Cancer 80:219, 1999
- gp100 presented by HLA-A2
Rosenberg SA, et al. Nat Med 4:321, 1998
Protein vaccine
Depends on cross-priming on autologous MHC molecules
Advantages
- non HLA restriction
- activation of both CD8+ and CD4+ T cells
Disadvantages
- Difficulty and expenses of generating recombinant proteins suitable
for human administration
Recombinant viral vaccines
Adenovirus, vaccinia virus, avipox
Mechanisms of action:
- Cellular damage, danger signals, cross-priming
- Direct infection of bone marrow derived APC
Disadvantages
- Neutralizing antibodies
- Previous exposure to cross-reacting viruses
- Previous immunization
Clinical trials
Weak generation of anti-tumor T cells
-Rosenberg SA, et al. J Natl Cancer Inst 90:1894, 1998 (Melanoma, MART-1 or gp100)
-Marshall JL, et al. J clin Oncol 23: 3963, 2000 (CEA)
- Eder JP, et al. Clin Cancer Res 5: 1632, 2000 (Prostate cancer, PSA)
Nucleic acid vaccines
Advantages
- easy to construct
- chemical stability
- inherently immunogenic, do not need adjuvants
- broad range of specific immune responses
- no presence of neutralizing antibodies
- less risk of insertional mutagenesis
- do not down-regulate MHC
Disadvantages
- Much less potent
- No replicative amplification
- Smaller inflammation
- No danger response
Heat shock proteins
gp96 and hsp70 purified from tumor cells
Mechanisms of action:
- Bind a wide array of peptides
- They introduce bound peptide into the MHC class I and II processing
pathways
- Binding of gp96 to macrophages induces secretion of proinflammatory
cytokines
Disadvantages
- Tumor tissue required
Clinical trials
- Belli, F. et al. J Clin Oncol 20:4169, 2002
Dendritic cells vaccines
Virus
Gene
Vector
AAA
AAA
AAA
AAA
Apoptotic bodies
Bacteria
Lysates
mRNA
Natural or
Synthetic peptides
Non genetic delivery
Genetic delivery
CD34 derived mature DC pulsed with several MHC class I
tumor peptides plus KLH and Flu-MP
Banchereau J, et al. Cancer Res. 61:6451, 2001
Mature monocyte-derived DC pulsed with several MHC class I
and class II tumor peptides plus TT
Schuler-Thurner, B et al. J Exp Med 10:1279, 2002
Thurner, B et al. J Exp Med 11:1669, 1999
Ongoing Phase I or II Nonrandomized Trials
of Cancer Vaccines
Ongoing Phase I, II, or III Randomized Trials
of Cancer Vaccines
What we have learn from clinical trials so far
- Vaccinations are safe and well tolerated
- No or transient major side effect (autoimmunity phenomena)
- Patients are immunized, with tumor specific T cell induction
or expansion
- Memory induction?
- Limited clinical benefits in heavily affected patients
Factors limiting the therapeutic impact of anti-tumor T cells
Lymphocytes factors
- CD4+ and CD8+ subsets
Tumor factors
- Production of immunosuppressive
cytokines
- Insufficient numbers, avidity
- Secretion of non appropriate
cytokines or not sufficient
lytic activity
- Regulatory T cells
- Loss of MHC molecules or tumor
antigens
Future challenges
- Best DC culture methods (maturation stage)
- Optimum antigen loading
- Most important TAA
- Vaccination schedule
- Dosages
- Route of injection
- Improvements/standardization of immunomonitoring
- Combination therapy
Strategies of antitumor immunotherapy
-Vaccination against tumor
neovascularization
Vaccination against tumor neovascularization
Preclinical studies
- DC pulsed with soluble VEGF-R2
- neutralizing antibodies
- CD8+ CTL
- Attenuated salmonella engineered
to express VEGF-R2
- CD8+ CTL