PhD SCIENCE REPORT AN M. T. VAN NUFFEL: Enhancing the costimulatory capacity of DCs by co-electroporation with CD40L, CD70 and/or constitutively active TLR4 mRNA

AN M. T. VAN NUFFEL

Despite the immunogenic nature of malignant melanoma and the reported induction of vaccine-induced cellular immune responses, limited clinical success has been achieved so far. The maturation status of the dendritic cells (DC) plays a pivotal role in their capacity to induce tumor-eradicating T cells. Recently, we have shown that so-called ‘TriMixDC’ have an improved T cell stimulatory capacity in vitro (Bonehill et al, Mol. Ther.). These TriMixDC are immature DCs (iDC) electroporated with mRNAs encoding CD40ligand (CD40L), CD70, a constitutive active form of TLR4 (caTLR4) and tumor associated antigen (TAA). Furthermore, in coöperation with Dr. N. Schaft and Dr. J. Dörrie, different mRNA constructs were compared for electroporation in DC: pGEM-plasmid derived, either enzymatically poly-adenylated or not, pST1-plasmid derived, or ‘Argos’ mRNA. The evaluated parameters were: he phenotypical and functional maturation of DC and their in vitro stimulatory capacity after electroporation with CD40L encoding mRNAs. Also the antigen presentation capacity after electroporation with different mRNAs encoding the tumor antigen MelanA was evaluated (manuscript in preparation). pST1-plasmid derived mRNA, containing two 3’ human ?-globin UTRs and a poly-A-tail of 120 adenines, without additional splice-site derived nucleotides at the very end, led to more and longer protein expression.

Following this, during 2008, the work was mainly focussed on the immunomonitoring of a pilot clinical study, set to investigate the in vivo stimulatory potential of TriMixDC in stage III/IV melanoma patients. Since November 2007, 17 patients (11M/6F, median age 49, range 29 – 62) received 4 biweekly autologous, TriMixDC vaccines of each ± 50 106 DC, injected intradermally. Each vaccine consists of gp100, tyrosinase, Mage-C2 and Mage-A3 antigen presenting TriMixDC which became mixed at equal ratios, aliquoted and cryopreserved until administration. Based on the results described above, all mRNAs used for transduction of patients-DC were pST1-plasmid derived. The tumor associated antigen (TAA) encoding mRNAs consisted of a fusion protein of the TAA with a class II sorting signal, DClamp.
The stimulatory capacity of DC was evaluated in blood by IFN? ELISPOT of pre- and post-vaccination PBMC pulsed with pools of 10 overlapping 15-mer peptides covering the whole sequence of the vaccinal TAA or MelanA. In 4 patients analysed so far, T cells specific for one or more vaccinal TAA were found pre-vaccination. Induction of T-cell responses was seen post vaccination. In addition, a ‘delayed type hypersensitivity’ (DTH) reaction was induced pre- and postvaccination. To quantify the percentage of activated TAA specific T cells, CD137 upregulation and cytokine secretion (IFN? and TNF?) were evaluated in response to autologous EBV-B cells expressing one of the vaccinal antigens or MelanA. When sufficient T cells were available, the cytolytic capacity (CD107a upregulation) of the CD8+ T cells was assessed and the region containing the recognized epitope was determined. In 13/17 patients TAA-specific DTH-infiltrating T cells were documented post-vaccination. Of these, 10/13 had a CD137+CD8+ T cell response and 6 of them responded to more than one TAA. We also observed CD4+ T cell responses in at least 2/13 patients. One of these patients mounted a CD4+ T cell response against multiple epitopes in addition to a CD8+ T cell response.

In the nearby future, more patients will become screened.
The PhD is predicted to end around august 2010.

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