Tumour immunotherapy

Tumour immunotherapy Raymond Steptoe Senior Research Fellow Diamantina Institute for Cancer, Immunology and Metabolic MedicineUniversity of Queensland, Princess Alexandra Hospital r.steptoe@uq.edu.au

Overview • History • Review of immune effectors & immunity • Basis for immunotherapy • Passive (Adoptive) immunotherapy • Active immunotherapy • Overview of a clinical trial (DC therapy)

History Immunotherapy: 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 tumour cell type specific (e.g) melanoma - MART-1/MelanA, tyrosinase , gp-100 B cell lymphoma - idiotype, CD20 AML - CD33 Prostate cancer – PSA, prostatic acid phosphatase shared (e.g.) MAGE-3 Carcinoembryonic 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 Cellular CD4+ 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 Molecular Cytokine -direct tumour killing (e.g. TNF-a) Antibody -coating of tumour cell – ADCC, CDC Macrophage Modified from: Schuster et al., Biotechnology J. 2006. 1:138-

Immune induction/effector pathways constitutive trafficking of dendritic cells NAÏVE T CELLS Priming and recirculation of effector T cells Modified from Banchereau et al., Ann Rev Immunol. 2000

Immune-escape of tumours - I • Immune inhibition • Inhibitory cytokines -TGF-b, IL-10, VEGF, -act on DC • Inhibitory signalling molecules • PD-1 ligands, NKIR • -act on T cells, NK cells • Inhibitory enzymes • -IDO, arginase • -act on T cells • T-cell inactivation • -dysfunctional DC • -chronic antigen stimulation x x x x Macrophage Modified from: Schuster et al., Biotechnology J. 2006. 1:138-

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 x x x x x Macrophage 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 cells Antibody

Passive (adoptive) immunotherapy Adoptive 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 mechanisms CD20 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 cancer VEGF 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) immunotherapy Adoptive 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 immunotherapy Adjunctive therapy -promotes immune responsiveness Immune-stimulatory cytokines -interleukin-2 (IL-2) -boosts function of T cells, NK cells -interferon-a2b (IFN-a2b ) -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 immunotherapy Vaccination (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 immunotherapy Vaccination (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 immunotherapy Vaccination (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

Overview Approved 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 Rituximab Gardasil (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-1b,TNF-a, PGE2) • Expansion from blood CD34+ stem cells GM-CSF/TNF-a TNF-a 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 loading in vitro maturation signal provided after antigen in vivo maturation signal provided along with antigen peptide pulsing(e.g. synthetic peptides, acid eluted) antibody-antigen conjugates(e.g. targeting endocytic receptors) MHC IMHC II whole-antigen pulsing(e.g. tumour lysate, recombinant protein) receptor-mediated uptake (e.g. mannosylated antigens) genetic-targeting (e.g. gene gun, gene therapy) genetic-targeting (e.g. RNA, DNA) helper epitopes boost CTL priming

DC immunotherapy using autologous tumour cell antigens at PAH Melanoma cell culture DC culture GM-CSF + IL-4 HBsAg Pulse DC

Administration / Migration of DC vaccines Routes Subcutaneous:-requires correct coordinated migration pattern Intranodal -direct admin to lymph nodes Dose? Injection of DC vaccine induces a systemic anti-tumour response

vaccine no vaccine Monitoring outcome Surrogate markers MHC-peptide tetramer ELISpot assay Enzyme-linked immunosorbent spot ICCS Intracellular cytokine staining Clinical markers Disease burden Clinical signs

Clinical Response: +ve HBsAg response Resp: PD: patient died, PR: partial remission, CR: complete remission

Clinical Response: no HBsAg response Resp: PD: patient died, PR: partial remission, CR: complete remission

Monitoring clinical outcome halo nevi Insert vitiligo vitiligo hair depigmentation

Monitoring clinical outcome ON ENTRY Pelvis Chest 4 MONTHS

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).

Tumour immunotherapy