Post on 28-Mar-2015
• Immunity and Tumors
• Cancer Immunotherapy
Immunity and Tumors
1890sColey treats patients with bacterial extracts
1950-1960sBurnet and Thomas- Immune-surveillance Hypothesis
“Major function of the immune system is to recognize and destroy arising malignantly transformed cells”
1957Prehn and Main- The origin of modern tumor immunology
Normal
Initiation
Promotion
Progression
proliferationdifferentiationapoptosis
proliferation
Pathogenesis of neoplasia
DNA damage(chemical, phisical, biologic) DNA damage
Clonal evolutionAdditional mutations
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’immunosorveglianzaThe 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 iltrapianto di tessuti normali viene accettatoIl rigetto di tumori spontanei o indotti conferisce protezione
2) Evidenze cliniche:L’insorgenza di tumori è piu’ alta in assenza di competenzaimmunologica (immunodeficienze congenite o acquisite)
Evidenze di immunogeneticità dei tumori
Sarcoma
Resezione
chirurgica
Nessuna crescita Crescita del tumore Nessuna crescita
Topo naive
Vaccinazione con cellule tumorali
Topo vaccinato
- 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
Conclusions from experiments on transplanted tumors
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
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
1) Inherited Immunodeficiency
Experiments in gene-knockout mice lacking various componentsof the immune system
- IFN-g deficient mice have a higher rate of both spontaneous and carcinogen- induced 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
Risposta primaria
Tumore (sorgente di antigene)
Cellule adibite alla presentazione antigenica
Linfociti T e B
Colocalizzazione
Elementi critici nello sviluppo di una risposta
anti-tumoraleRisposta secondaria
Tumore
Cellule adibite alla presentazione antigenica
Linfociti T e B
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
Tumor-specific immune responses
Tumor-specific T cellsBlood
Transforming eventLymphBlood
Tumor antigens or tumor cells
Immune stimulation or inflammation
Spontaneous inflammation in the tumor
microenvironment
Antigen-specific immunization
Non specific immune
stimulation
Tumor growth
Tumor regressio
n
Tumor progressio
n
The fine balance between immune responsiveness and immune resistance
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
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
Main lymphocytes subsets participating to anti-tumor responses
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, EGAnnu 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
Effector phaseCD4+CTL
CD8+CTL
Immaturedendritic cell
Tumor cell
DrainingLymph node
Tumor cell Macrophage
MHCClass II
MHCClass I
Tumor antigens
Tumor peptides
Release ofgranule contents
Maturedendritic cell
CD8+ T cell
CD4+ T cell
Killing
CD40
CD40L
Killing
Reactive oxigenintermediates
Th2
Th1
Th1
B cellTh1/Th2
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 immunityIgnorance
-Tolerance inductionAnergy/Deletion
- Mechanisms of immune escape
Tumor cell
T cell
Tolerance
Antigen uptake bytolerance-inducing APC
T cell
APC
Failure to develop efficient anti-tumor immunity
TCR
MHC-peptide
Receptor for costimulatorysignals
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 - Active vaccination
- Monoclonal antibodies
- 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 tumorinfiltrating lymphocytes (TILs)
Adoptive transfer of TILs expanded in vitro and high dose IL-2 following a non-myeloablative conditioning regimen
Dudley ME et al. Science, 2002
Antigen specific T cells transfer
Adoptive transfer of antigen-specific CD8+ T cell clones
Yee, C. et al. PNAS 99: 16168, 2002
In vivo persistence, migration and antitumor effect of transferred MART-1/Melan-A specific T cell
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
- Choice of the antigen
Undefined (cancer cell extracts, mRNA)
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-A2Rosenberg 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 vaccinesAdenovirus, 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 trialsWeak 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
AAA
AAAAAAAAAApoptotic bodies
Lysates mRNABacteria
Natural orSynthetic peptides
Gene
Virus
Vector
Genetic deliveryNon genetic delivery
Banchereau J, et al. Cancer Res. 61:6451, 2001
CD34 derived mature DC pulsed with several MHC class Itumor peptides plus KLH and Flu-MP
Thurner, B et al. J Exp Med 11:1669, 1999
Schuler-Thurner, B et al. J Exp Med 10:1279, 2002
Mature monocyte-derived DC pulsed with several MHC class I and class II tumor peptides plus TT
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
- Insufficient numbers, avidity
- Secretion of non appropriate cytokines or not sufficient lytic activity
- Regulatory T cells
Tumor factors
- Production of immunosuppressive cytokines
- 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
- DC pulsed with soluble VEGF-R2 - neutralizing antibodies - CD8+ CTL
- Attenuated salmonella engineered to express VEGF-R2 - CD8+ CTL
Preclinical studies