Ingen lysbildetittel

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mRNA transfection of dendritic cells Gustav
Gaudernack
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Strategies for immunotherapy
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mRNA transfected DCs as vaccines what kind of
antigen

• Two approaches (general and individualized)
• mRNA encoding defined tumour antigens (PSA,
  hTERT, survivin etc)
• mRNA from whole tumour

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Why tumour mRNA?
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Molecular portraits of human breast
tumours nature 17 Aug. 2000
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Approach to mRNA vaccination taken at
Rikshospitalet-Radiumhospitalet HF

• Hypothesis Highly expressed and aberrantly
  expressed mRNAs may encode tumour antigens
• To go from tumour mRNA profiling to
  individualized vaccination against multipple
  antigens
• Target all potential antigens (autologous tumour
  mRNA)
• Target shared expression profile antigens (tumour
  cell line mRNA)

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General protocol for vaccination cancer patients
with own dendritic cells transfekted with whole
tumour mRNA
Tumor celle lines or fresh tumour tissue
Leukapheresis
Elektro-poration
Monocytes GM-CSF, IL-4
mRNA
Cancer vaccine
SSL/GG 2000
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Development strategy and aims

• Pre clinical development
• Large scale GMP production
• Efficient antigen transfer
• Generation of T cell responses against mRNA
  transfected DC

• Aims
• Initiate clinical trials
• Optimize design of clinical protocols
• Define target patient groups for future trials
• Define new tumour antigens recognized by T cells
  and antibodies

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DC culture

• Day - 7 CellGro DC medium
• 2500 U/ml GM-CSF 1000U/ml
  IL-4
• Cell concent. in the bags 1x
  106/ml
• Day - 2 Transfection with tumour mRNA

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TEFLON BAGS
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DC culture

• Day - 2 Following transfection of tumour
  mRNA
• Maturation of DC by 10ng/ml
  IL- 1b, 10ng/ml TNF-?, 1000U/ml IL-6, 1ug/ml
  Pge2, 2500 U/ml GM-CSF 1000U/ml IL-4
• Day 0 Harvesting and freezing

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CD 1a
CD 83
Immature DC
Mature, transfected DC
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Maturation associated markers
Immature DC
Mature, transfected DC
CCR7
CD209
CD208
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EGFP mRNA transfection of immature DC by
electroporation
A Control
Propidium iodide(92)
BTransfected
Propidium iodide(86)
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GFP expression after DC transfection with GFP mRNA
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Standard conditions Allows 1-2 x 100.000.000
cells/transfection
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Transfection of DCs in the mature state
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All cells
CD4
CD8
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Room for technical improvement?

• Documentation of transfection efficacy spike in
  hTERT mRNA gt quality assurance by TRAP assay or
  Real Time PCR in cellular product
• Documentation of T cell reactivity against known
  tumour antigens use DC transfected with hTERT
  mRNA only as stimulators/targets in immune
  monitoring
• Added bonus T cell responses directed against
  telomerase

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Real Time PCR DC transfection with hTERT-mRNA
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TRAP-assay Telomerase activity after DC
transfection with hTERT-mRNA or melanoma-RNA
Preclinical full scale evaluation, Cancer Gene
Therapy (2005)
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Ongoing clinical trials
Phase I/II trial of immuno gene therapy with
mRNA- transfected dendritic cells in pediatric
leukemia patients up to 10 patients Phase I/II
trial of immuno gene therapy with mRNA-
transfected dendritic cells in pediatric patients
with solid tumours (neuroblastoma, brain tumours,
sarcomas) up to 10 patients. Two under inclusion
(brain tum.) Phase I/II trial of immuno gene
therapy with mRNA- transfected dendritic cells
after Treg depletion in patients with metastatic
malignant melanomas (Stage III) 20 patients.
Three patients included.  
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mRNA dose and survival
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Plans for RNA/DC trials

• Intradermal vaccination
• Patients with less advanced disease (safety OK)
• Counter CD4CD25 regulatory T cells
• IL2-toxin conjugate (Dannull et.al., J Clin
  Invest 2005)
• anti-CTLA4 antibody (Ribas et al, JCO 2005)
• transfection of DCs with siRNA for IL-10/TGF-b
  (M. Sioud)
• chemotherapy immunotherapy (Gaudernack, ongoing
  trial)
• Upcoming DC-vaccine trials (RR Montebello)
• Melanoma (stage III) RNA/DC chemotherapy
• prostate cancer tumor-RNA hTERT
• ovarian carcinoma amplified tumor-mRNA

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