Gene therapy of lung cancer Where we are and where to go?

Gene therapy of lung cancer Where we are and where to go?

Gene therapy of lung cancer Where we are and where to go?

GT in TUMORS: TARGETSTumor cellsImmune systemHematopoietic stem cells (HSCs)

Target 1: Tumor cell

Target 2: Immune system

Transferring genes conferring their resistance to high dose chemotherapy (HDC) into hematopoietic stem cells Supramaximal chemotherapy therapy possible in clinical trials concerning breastovarytesticlesmall cell lung cancer because of specific drug prophile 1) ABC proteins (as MDR-1) 2) genes of antioxidant enzymes (GS-transferase) are consideredTarget 3: HSCs

Target 1: Tumor cell

Supplementary (Substitutive) GTClinical trial examples

Development(clinical phase)GeneApplicationIIIP53 (Ad5CMV-p53) Planoepithelial cancer of head & neck, NSCLCIIIAllovectin (HLA-B7/microglobulin 2)Advanced melanoma (adiuvant for chemiotherapy) IIITNF- (TNFerade)Pancreatic cancer (adiuvant for chemiotherapy) I, IICycline GMetastases of colon cancer to liverIIIL-2Transfer of autologous lymphocytes in melanoma metastases

Suppressory GT of tumorXXXTriple helix target: DNAAntisens, siRNA, target: mRNAAptamers, target: protein IGF-I, survivinePKC, clusterin, BCL-2, c-erbB2, EGF, VEGFVEGF, MUC1,

Antisense therapy 1 triple-helix anti-IGF-IFirst strand is a transcript of pMT AG vector (homopurine RNA); 2nd and 3rd strand represent typical genomic DNA (promoter of IGF-I gene)I I I I I Watsona-Crick bonds****** Hoogsteen bonds

Suppressory therapy 1 Clinical applications of Antisense ON

DevelopmentTargetApplicationI, IIIBcl-2 (Genasense)SC lung cancer; melanoma, myeloma multiplexI,II,IIIProtein Kinase C-alpha (ISIS 3521)NSC lung cancer, metastases of colon cancer, prostate cancer (HR)IIC-raf Kinase (ISIS 5132)metastases of colon cancer; prostate cancer (HR), lung cancerIC-myb Chronic myeloid leukemiaIH-ras (ISIS 2503)Breast cancer

Suppressory therapy 1 Clinical applications of Antisense ON

DevelopmentTargetApplicationI, IIIBcl-2 (Genasense)SC lung cancer; melanoma, myeloma multiplexI,II,IIIProtein Kinase C-alpha (ISIS 3521)NSC lung cancer, metastases of colon cancer, prostate cancer (HR)IIC-raf Kinase (ISIS 5132)metastases of colon cancer; prostate cancer (HR), lung cancerIC-myb Chronic myeloid leukemiaIH-ras (ISIS 2503)Breast cancer

Suppressory therapy 2 Clinical applications of Antisense ON

DevelopmentTargetApplicationI-IIIBcl-2 (Genasense, SPC2996)Melanoma, CLL (adiuvant for chemiotherapy) I, IIX-IAP (AEG35156-103)Recurrent AML, breast cancer, NSCLC, different type tumor dissemination (adiuvant for chemiotherapy) IIClusterin (OGX-011) Neo-adiuvant for therapy of prostate cancerIHIF-1 (EZN-2968)Different tumors

Suppressory therapy 3 Ribozymes (bcr-abl; ras)

Native siRNA, products of nucleolytic dsRNA degradation driven by ribonuclease Dicer have charakteristic composition.,, siRNA features2nt unpaired 2nt unpaired

RNAi phases

I siRNA duplex generation IIRISC silencing complexformationIIIRISC activation and recognition of mRNA complementary to antisense siRNA strandIVFinal mRNA degradation

Consequence:Gene silencing (no protein expression)

Limitations of RNAi RNAi efficiency is limited by ability of synthetic siRNA molecules to bind mRNA and inhibit the target gene. Different siRNA molecules directed against the same gene fragment are characterized by different silencing potential. Thats why efficiency of each siRNA type should be evaluated separately.

It was also found, that RNAi specifity depends on the amount of siRNA used in the assay. Its excess induces expression of many genes associated with cell response to the stress / viral RNA.

SELEX

Apoptosis

TRAIL receptors activation

Apoptosis

Inhibition of anti-apoptotic Bcl-2 protein family

Inhibition of anti-apoptotic Bcl-2 protein family

Inhibition of tumor cell proliferationPROBLEMSWe need to reach and kill all tumor cellsThe problems exist to gain solid tumor interior tissueContinous oligonucleotide infusion is difficult to provideAlternative plasmid in vivo therapy is limited due toLow transfection efficacyLack of lung cancer specific promoterLow transcription level

*

Safety Study of an Antisense Product in Prostate, Ovarian, NSCLC, Breast or Bladder Cancer (OncoGenex Technologies, USA)

Phase IIOGX-427 is a second-generation ASO that inhibits expression of Hsp27. Hsp27 increases with cell stress, cytotoxic chemotherapy, radiation therapy and hormone therapy and has been shown to inhibit cell death. Patients (major inclusion criteria): >18 years; metastasesGX-427 injections at 200mg, 400mg, 600mg, 800mg or 1000mg once a week until withdrawnDrug: Docetaxel

Angiogenesis: VEGF as GT targetEarly effectsLate effectsRegression of small vessels growthVascularisation inhibition

Bevacizumab binds VEGFVEGFR-2VEGFR-1Endothelial CellVEGFAnti-VEGF antibody(Bevacizumab)

Anti-VEGF Strategies

Target 2: Immune system

TUMOR CELLIrradiated tumor cellsPlasmidsDendritic cellsPeptidesViral vectorscoding neo-antigens

TUMOR CELLIrradiated tumor cellsPlasmidsDendritic cellsPeptidesViral vectorscoding neo-antigens

TUMOR CELLIrradiated tumor cellsPlasmidsDendritic cellsPeptidesViral vectorscoding neo-antigens

Specific ways to generate dendritic cells (DC)

Immune response against tumorsFrequent lymphocyte infiltrates (Tumor infiltrating lymphocytes, TILs) in tumor tissue TIL reveal phenotype of cytotoxic effector cells, they are able to kill tumoer cells It has been demonstrated that TIL cells present specific receptors (TCR) against known neo-antigens (usually tumor associated antigens, TAA)Lung cancer antigens of determined amonoacide sequence: MUC1MAGE-A3EGFRTGF-2hTERT

Antigen (TAA, TSA)TCRMHC I / MHC II CD8 / CD4CD80 / CD86 CD28Fas (CD95) FasL (CD178)ICAM-1 (-2) LFA-1LFA-3 CD2CD40 CD40LDendritic cell vs naive T (CD4 or CD8) lymphocyte

Antigen (TAA, TSA)TCRMHC I / MHC II CD8 / CD4CD80 / CD86 CD28Fas (CD95) FasL (CD178)ICAM-1 (-2) LFA-1LFA-3 CD2CD40 CD40L1Dendritic cell vs naive T (CD4 or CD8) lymphocyte

Antigen (TAA, TSA)TCRMHC I / MHC II CD8 / CD4CD80 / CD86 CD28Fas (CD95) FasL (CD178)ICAM-1 (-2) LFA-1LFA-3 CD2CD40 CD40L12Dendritic cell vs naive T (CD4 or CD8) lymphocyte

Antigen (TAA, TSA)TCRMHC I / MHC II CD8 / CD4CD80 / CD86 CD28Fas (CD95) FasL (CD178)ICAM-1 (-2) LFA-1LFA-3 CD2CD40 CD40L123CYTOKINES, e.g. IL-2Dendritic cell vs naive T (CD4 or CD8) lymphocyte

Antigen (TAA, TSA) TCRMHC I / MHC II CD8 / CD4CD80 / CD86 CD28Fas (CD95) Fas Ligand (CD178)ICAM-1 (-2) LFA-1LFA-3 CD2CD40 CD40LTumor cell vs primed T (CD4 or CD8) lymphocyte

Low or altered expression of TAA antigens on tumor cells Decreased or absent MHC I molecules Atypic MHC II molecules (?)Lack of B7 costimulatory moleculesLack of adhesive molecules as LFA-1,-3 or ICAM-1Superficial anti-adhesive molecules (mucin) on tumor cellsIMMUNE TOLERANCE IN TUMORS 1

IMMUNE TOLERANCE IN TUMORS 2 Immunosupressive factors secretion: TGF-beta, IGF-I L-6, IL-10, IL-13PGsTumor cells are not susceptible to cytotoxic activity of cytokines, as for example soluble FasL or TNFs Proapoptotic molecules (FasL) on tumor cells wich can kill TILsActive tumor participation in local inflammatory cell/ mediator network in order to modify Th1/Th2 ballance towards Th2 domination; improve cancer cell survival inter alia with use of proinflammatory cytokines (e.g. TNF- of anti-tumor immunity versus TNF receptor type 2);Reprogramme DCs towards immune tolerance of TAA (regulatory T cells)

Heterogenous solid tumorMetastases generationPrzerzutPrzerzutPrzerzutTUMOR Progressionmetastasismetastasismetastasis

DNA Vaccines 1

Phase of clinicla trialCommercial nameTherapeutic tool/ Notes II1650-GAllogenic NSCLC cellsIIAd-CCL23MoDC modified with AdV vector encoding CCL23 (chemokine with strong T cell activation properties), administered directly to the tumorIIMelCancer VacMoDC fused (Hybridoma) with melanoma cells (immunogenic, neo-antigens cross-reactivity with lung cancer ) Advanced non-operative NSCLC + Cox inhibitors+ IL-2IIINGN-225MoDC modified with AdV encoding mutant p53 (as mutated it serves as neo-antigen )

*Carcinoembryonal antigen**TRICOM = B7.1 (CD80) + ICAM-1 + LFA-3 DNA Vaccines 2

Phase of clinicla trialCommercial nameTherapeutic tool/ Notes IIIBLP-25/ STARTVaccination with MUC1 antigen in liposomal career, MUC1 expression on lung cells is necessary, significant increase in 3 years survivalIIITG4010Avian vaccinia virus vector, transgene: MUC1 + IL2 ; MUC1 expression on lung cells is necessary,IFowlpox-CEA/ TRICOMAutologous MoDC transfected with Avian vaccinia virus vector; transgene: TAA (CEA*) + TRICOM**Different tumors, including lung cancer; CEA expression on tumors cells necessary

Target 2: Immune system

Target 2: Immune system

Generations of CARs

TAA-specific TCR receptors & Chimeric CAR receptors (Acc. Nature Reviews/Immunology 2011, zmodyfikowane)

Note: TAA antigen sequence must be defined

Limitations of GT 1Short term character of GTDNA and/or RNA introduced to cells must be active for a longer time Transfected cells should live and function normally However, in supplementation GT transgene integrates with genome with difficulties; the site of integration is completely unpredictableCell divisions often eliminate effects of transfection - patients need the repetitive therapies

Limitations of GT 2Immune response

In GT in vivo patient exposition to foreign antigens (DNA, protein career) may induce immune reaction In supplementation GT, immune system response is more and increased with consecutive trangene exposition

Limitations of GT 3Troubles with viral vectorsShortcomings:toxicity, Immune and inflammatory response, Infection of unwanted cells,problems with control of transgene expression.

There is a a risk of virus pathogenic features recovery, despite assumed defective character of wirus used for vector contruction (onkogenesis not excluded!).

Limitations of GT 4Multifactor ethiopathogenesis of diseases

In some cases only, the disease could be cured with approach directed again one single geneGT systems based on multi-gene simultaneous correction in the same cell are, up to date, not sufficient enough

Two individual patient storiesAshanti da Silva / Jessi Gelsinger

Where are we?Perspectives of tumor GTClinical trials by phase

2010: only circa 30 trials directed against tumors reached the phase IIIDelivery of immune stimulating cytokines or anti-cancer genetic vaccines

Where we go?Perspectives of GT in tumorsGene Therapy + Immune therapy = immune gene therapy?GT vaccinations as adjuvant therapy?GT as chemotherapy assistance?go in all? Improved vectors and transfection systems?Can we really mimic full succesful anti-tumor immune responses? Turning point or step by step progress?

Thank you for your attention

***************Antisense nucleotide bcl-2 mRNA degradation no protein translation*Antisense nucleotide bcl-2 mRNA degradation no protein translation**Presta et al. Cancer Res. 1997;57:4593.VEGF is a key regulator of tumor angiogenesis.Angiogenesis, the growth of blood vessels from existing vessels, is an important feature of tumor growth.Avastin is a recombinant humanized monoclonal antibody that binds VEGF.Avastin binds to VEGF with high specificity and affinity resulting in potent VEGF-neutralizing activity.Once bound, Avastin prevents VEGF interactions with VEGFR-1 (Flt-1) and VEGFR-2 (KDR) on the surface of endothelial cells, inhibiting VEGF-stimulated downstream signaling events.****************