Raymond SteptoeRaymond SteptoeSenior Research Fellow Senior Research Fellow

Diamantina Institute for Cancer, Immunology and Metabolic Diamantina Institute for Cancer, Immunology and Metabolic MedicineMedicine

University of Queensland, Princess Alexandra Hospital University of Queensland, Princess Alexandra Hospital

r.steptoe@uq.edu.aur.steptoe@uq.edu.au

Tumour Tumour immunotherapyimmunotherapy

Overview

• History

• Review of immune effectors & immunity

• Basis for immunotherapy

• Passive (Adoptive) immunotherapy

• Active immunotherapy

• Overview of a clinical trial (DC therapy)

HistoryImmunotherapy: actively enhance immune response or passively

deliver immune effectors

1890’s Coley’s toxins ( streptococcus/staphylococcus)

Surgical intervention

Radiotherapy

Chemotherapy

1960’s – 70’s Understanding of role of immune system in animal models

1980’s Mixes of tumour cells and bacteria (e.g BCG) Cytokines: IFN-alpha, interleukin 2 Adoptive transfer of in vitro activated T cells

1990’s Peptide and recombinant antigen vaccines Gene-engineered tumour cell vaccines Dendritic cell vaccines

Why immunotherapy?Rationale is based on:

• evidence from mouse models -immune-compromised mice have increased incidence of cancers-immunisation induces tumour-specific immunity & reduces tumour mass/tumour growth

• clinical observations -spontaneous regressions-immunodeficiency increases some cancers-immune infiltrates –better prognosis-tumour specific T cells can be isolated

Use effectors of the immune system to kill tumours

Tumour antigens -immune targets Tumours are ‘altered self’

Tumour antigens are usually self-proteins selectively over expressed by a tumourcell type specific (e.g)

melanoma - MART-1/MelanA, tyrosinase , gp-100B cell lymphoma - idiotype, CD20AML - CD33Prostate cancer – PSA, prostatic acid phosphatase

shared (e.g.)MAGE-3Carcinoembryonic antigen (CEA)HER2/neu

Peptides defined from molecular approaches

Whole proteins defined by responses in tumour-bearing individuals

Non-selective methods (eluted peptides, fusions)

Effectors of the immune system

Modified from: Schuster et al., Biotechnology J. 2006. 1:138-

CellularCD4+ T cell-produces cytokines-helps for CD8+ T cells and B cells

CD8+ T cell (CTL)-direct lysis/killing of antigen-expressing cells

B cell -produces antibody (Ab)

Granulocyte- Ab-dependent cytotoxicity

Macrophage-cytokine-induced killing-Ab-dependent cytotoxicity

Natural killer cell-direct lysis of tumour cell target-Ab-dependent cytotoxicity

MolecularCytokine-direct tumour killing (e.g. TNF-)Antibody-coating of tumour cell – ADCC, CDC

Macrophage

Immune induction/effector pathways

Modified from Banchereau et al., Ann Rev Immunol. 2000

constitutive traffickingof dendritic cells

NAÏVE T CELLS

Priming and recirculation of effector T cells

Immune-escape of tumours - I

Immune inhibitionInhibitory cytokines -TGF-, IL-10, VEGF, -act on DCInhibitory signalling molecules-PD-1 ligands, NKIR

-act on T cells, NK cellsInhibitory enzymes

-IDO, arginase-act on T cells

T-cell inactivation -dysfunctional DC-chronic antigen stimulation Macrophage

xxx

x

Modified from: Schuster et al., Biotechnology J. 2006. 1:138-

Macrophage

xx

xxx

Immune-escape of tumours - II

Antigenic loss variants

Loss/down regulation of antigen targets -Tumour Specific Antigens

-loss of CD4+ & CD8+ T-cell epitopes -CD20

-loss of antibody binding (Rituximab)

Loss of MHC class I / antigen processing -MHC class I expression -TAP etc. (for processing /loading)

- loss of CD8+ T cell recognition

Modified from: Schuster et al., Biotechnology J. 2006. 1:138-

Immunotherapy - purpose

• actively enhance immune response or passively deliver immune effectors

-boost impaired components

-replace missing elements

Passive (adoptive) immunotherapy

Transfer of efferent elements of the immune system

Effector T cells in-vitro activated T cells

Antibodies -surface antigens CD20 Non-Hodgkins lymphoma -growth factors / receptors HER2/neu breast cancer VEGF colorectal cancer

Macrophage

Effector T cellsAntibody

Passive (adoptive) immunotherapyAdoptive antibody therapy - targets surface molecules expressed or over-expressed by tumour cells

Antibody-dependent cytoxicity (ADCC), complement-dependent cytotoxicity (CDC) – cells are killed by these mechanismsCD20 Rituximab Non-Hodgkins lymphoma (NHL)CD33 Gemtuzumab Acute myelogenous leukemia (AML)CD52 Alemtuzumab Chronic lymphocytic leukemia (CLL)

Disruption of signalling through receptors or growth factors -prevents growth of cells

HER2/neu Herceptin Breast cancerVEGF Avastin Colorectal cancer (CRC)EGF-R Cetuximab Colorectal cancer (CRC)

-limitations-loss of antigen expression- large quantities required/expensive- surface molecules only – limits repertoire

Passive (adoptive) immunotherapyAdoptive cellular therapy (ACT) -provides an exogenous source of anti-tumour T cells

Patient’s own T cells are activated in vitro and retransferred -tumour specificity generated by:

-using defined tumour-specific antigen-tumour infiltrating lymphocytes

-most effective for highly immunogenic tumours-melanoma-EBV-induced post-transplant lymphoproliferative disorder-allogeneic HSCT for acute myelogenous leukemia

-may be boosted by concurrent immunisation etc.

-can target intracellular proteins, more diverse targets than antibody

-limitations-persistence of transferred cells

(overcome by lymphodepletion)-diverse specificities required-experimental procedure

Rosenberg et al. Nat Rev Cancer, 2008

Active immunotherapyAdjunctive therapy-promotes immune responsiveness

Immune-stimulatory cytokines-interleukin-2 (IL-2)

-boosts function of T cells, NK cells -interferon-2b (IFN-2b )

-mechanism unclear Limitations:

-side effects-limited effectiveness

Suppression of immune inhibitors-lymphodepletion (promotes expansion of antigen-specific T cells)-anti-CTLA4 (prevents inactivation of T cells)-anti-PD-L1 (prevents inactivation of T cells)

-limitations:

-experimental

Active immunotherapyVaccination (therapeutic) -boosts ‘ineffective’ T cell responses

Whole tumour vaccines

-tumour cells poorly immunogenic so immunogencity must be increased-addition of BCG-addition of adjuvants-use of allogeneic tumour cells -gene-engineering of tumour cells -cytokines-GM-CSF,

-costimulatory molecules B7

-evidence of T-cell priming often apparent in vitro, but with little clinical effect

-limitations-modest clinical effects-under development

Active immunotherapyVaccination (therapeutic) -boosts ‘ineffective’ T cell responses (and induces de-novo responses?)

Specific antigen vaccines -a range of tumour-specific antigens have now been defined (see Kim et al., - best prospects are those that are widely expressed in tumours

-synthetic peptide fragments-recombinant proteins -DNA/RNA

- delivery vectors -’conventional’ adjuvants- viral delivery-dendritic cells

-evidence of T-cell priming often apparent in vitro, but with little clinical effect

-limitations-modest clinical effects-under development

Active immunotherapyVaccination (prophylactic) -primes responses in a ‘naïve’ immune system and generates protective immunity

-limited applications

Gardasil (Merck) & Cervarix (GSK) – cervical cancer -exploits known features of the human papilloma virus life-cycle.

HPV-induced cervical cancer requires in infection with HPV (6,11,16,18 etc.)

-immunisation with virus-like particle containing HPV E6, E7 induces strong neutralising antibody responses and prevents HPV infection.

-limitations-not all pathogenic HPV serotypes targeted-cancer must be virally induced

OverviewApproved immunotherapies primarily passive strategies

Development of active immunotherapy has been slow-limited somewhat by stage of disease treated (ie late/advanced disease)

Immunotherapy (primarily) is considered an adjunct to ‘conventional’ therapies and particularly for clearing minimal residual disease of metastases

Noteable success have been very profound RituximabGardasil (Cervarix)

DC vaccines

Overview of a clinical trial

Considerations for DC vaccines

• Generation

• Antigen loading / maturation

• Administration

• Migration

• T-cell activation

• Monitoring: clinical markers/surrogate markers

• Quality control

Generation of DC for vaccine use• In-vitro conversion of monocytes

GM-CSF/IL-4 + maturation cocktail (IL-6, IL-1,TNF-, PGE2)

• Expansion from blood CD34+ stem cellsGM-CSF/TNF-

TNF- matures DC

labour intensive, expensive, QC issues

• Harvest from blood with / without growth factor-induced mobilisation

maturation procedure

limiting cell number obtained

DC are generated from each individual (because of MHC differences between individuals)

Antigen loadingin vitro

in vivo

peptide pulsing(e.g. synthetic peptides,

acid eluted)

whole-antigen pulsing(e.g. tumour lysate,

recombinant protein)

genetic-targeting (e.g. RNA, DNA)

genetic-targeting (e.g. gene gun, gene therapy)

antibody-antigen conjugates(e.g. targeting endocytic receptors)

receptor-mediated uptake(e.g. mannosylated antigens)

MHC IMHC II

helper epitopesboost CTL priming

maturation signal provided along

with antigen

maturation signal provided after

antigen

DC immunotherapy using autologous tumour cell antigens at PAH

Melanoma cell culture

DC culture

GM-CSF + IL-4

HBsAgPulse DC

Administration / Migration of DC vaccines

RoutesSubcutaneous:-requires correct coordinated

migration pattern

Intranodal -direct admin to lymph nodes

Dose?

Injection of DC vaccine induces a systemic anti-tumour response

Monitoring outcome

no vaccinevaccine

ELISpot assayEnzyme-linked immunosorbent spot

MHC-peptide tetramerSurrogate markers

ICCSIntracellular cytokine staining

Clinical markersDisease burdenClinical signs

Clinical Response: +ve HBsAg response

Age/sex Site Dose (DCx106/kg)

Vaccines Resp Duration (months)

35F Liver, s.c. 0.05 5 PD - 67M skin 0.02 5 PD - 50M s.c., adrenal, LN 0.05 8 PR 3 40M s.c 0.07 8 CR 29+ 62M s.c., spleen, LN 0.09 8 PD - 75F Pelvis, lung 0.09 8 PR 8 48F s.c. 0.11 7 PD - 45F s.c. 0.07 8 PD - 60M Lung, s.c. 0.07 8 SD 2+ 39F Pelvis, LN, bowel 0.07 7 PD -

Resp: PD: patient died, PR: partial remission, CR: complete remission

Clinical Response: no HBsAg response

Age/sex Site Dose (DCx106/kg)

Vaccines Resp Duration (months)

41F LN 0.02 8 PD - 37F Abdomen, s.c.,

adrenal, spleen, lung 0.02 3 PD -

66M s.c. 0.02 3 PD - 43M s.c., brain 0.05 8 PD - 57F Liver, abdomen,

mediast., s.c., bone 0.07 4 PD -

52F Liver, lung 0.11 6 PD - 72M s.c. 0.07 5 PD - 44M Lung, spleen, s.c. 0.07 8 PD - 63M Adrenal, lung 0.07 8 PD - 71M Lung 0.07 5 PD - 31F LN, bowel 0.07 5 PD - 33M LN, lung 0.07 5 PD -

Resp: PD: patient died, PR: partial remission, CR: complete remission

Monitoring clinical outcome

Insert vitiligo

halo nevi

vitiligo hair depigmentation

Monitoring clinical outcome

ON ENTRY

4 MONTHS

Pelvis Chest

Overview- DC therapy

DC therapy :

Safety – very good

Outcome - not 100% effective (~ 20%) -disease and stage-dependent

Key points to remember

‘Approved’ immunotherapies primarily passive strategies

Development of active immunotherapy has been slow -still experimental

- large range of tumour-specific antigens defined for some tumours

-limited somewhat by stage of disease treated (ie late/advanced disease)

Immunotherapy (primarily) is considered an adjunct to ‘conventional’ therapies and particularly for clearing minimal residual disease of metastases

Noteable success have been very profound

Rituximab

Gardasil

Further reading

• Immunotherapy of melanoma:Fang et al., Journal of Investigative Dermatology, 128:2596- (2008).Kirkwood et al., Journal of Clinical Oncology, 26:3445- (2008).

• Adoptive T cell therapy:Rosenberg et al., Nature Reviews Cancer, 8:299-, (2008).

• DC therapy:Banchereau & Palucka, Nature Reviews Immunology, 5:296-, (2005).