• 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