Preface

1 Introduction

2 Material and Methods

• 2.1 FKN gene expression in NXS2 cells and neuroblastoma tissue was demonstrated by RT-PCR.

• 2.2 FKN protein expression in vitro and in vivo

• 2.3 Determination of the chemotactic activity of FKN expressed by NXS2 cells in vitro and in vivo

  • 2.3.1 Migration assay

  • 2.3.2 Immunohistochemistry

• 2.4 In vivo depletion of CD4⁺ and CD8⁺ T lymphocytes

• 2.5 Cytotoxicity assay

• 2.6 Flow cytometry

• 2.7 Statistics

3 Results

• 3.1 Construction of a mammalian expression vector encoding mFKN

• 3.2 Confirmation of the gene transcription and protein expression of mFKN in neuroblastoma cells and primary tumors

• 3.3 Determination of the chemotactic activity of FKN produced by NXS2 cells in vitro and in vivo

• 3.4 Effect of targeted IL-2 with ch14.18-IL-2 on FKN gene therapy

• 3.5 Tumor-specific CTL activity of mice following FKN and ch14.18-IL-2 combination therapy

• 3.6 Upregulation of T cell activation markers and pro-inflammatory cytokines following FKN gene therapy and targeted IL-2

• 3.7 Role of CD4⁺ and CD8⁺ T cells in tumor inhibition by FKN gene therapy and targeted IL-2

4 Discussion

Abkürzungsverzeichnis

Literaturverzeichnis

Anteilserklärung

Acknowledgment

Eidestattliche Erklärung

Fig 1. Schematic structure of FKN (4)

Fig. 2 Strategy of FKN gene therapy combined with targeted IL-2.

Fig. 3 Detection of FKN gene expression in NXS2 cell lines. NXS2 cells were stably transfected with a plasmid encoding for FKN (NXS2-FKN) as previously described [4] and subjected to gene expression analyzed by RT-PCR. Results were compared to NXS2 wildtype and NXS2 mock transfected control groups. The presence of a band at 1.1 kb indicates the expression of FKN. GAPDH was amplified as an internal control (0.3 kb). M: 100 bp ladder, Invitrogen. 1: NXS2 wildtype cells. 2: NXS2 mock transfected. 3: NXS2-FKN.

Fig. 4 Determination of the FKN protein expression by NXS2 cells. The FKN protein expression as a secreted and a membrane bound protein was determined by sandwich ELISA in the supernatant of cultured cells (A) and by flow cytometry on the cell surface (B). (A) The production of soluble FKN protein was quantified by a commercial ELISA in culture supernatants of 10⁶ cells after 24h. Results indicate MV ± SD of FKN secretion rates in ng/ml/24h obtained from triplicate experiments. 1: NXS2-FKN 3rd generation subclone, 2: NXS2-FKN bulk culture, 3: NXS2 mock transfected cells, 4: NXS2 wildtype cells. Asterisks indicate non-detectable levels of FKN. (B) The presence of the membrane bound FKN protein was quantified by flow cytometry. Black: NXS2 wild type cells, yellow: NXS2 mock transfected cells, green: NXS2-FKN bulk culture, red: NXS2-FKN 3rd generation subclone.

Fig. 5 FKN mediated chemotaxis in vitro. FKN mediated chemotaxis was determined in a boyden chamber assay using 2 x 10⁵ splenocytes (37°C, 5% CO₂, 6h). The total number of transmigrated cells was determined microscopically. The data are expressed as percent of transmigrated cells and represent MV ± SD of triplicate experiments. 1: serum free NXS2-mock supernatant (negative control), 2: recombinant murine FKN 12,5 ng/ml, 3: 20 ng/ml, 4: 50 ng/ml, 5: serum free NXS2-FKN supernatant, 6: serum free NXS2-FKN supernatant plus 2 µg/ml anti-mFKN mAb (M18).

Fig. 6 Analysis of tumor infiltrating lymphocytes following FKN immunogenetherapy. The chemotactic activity of FKN produced locally in the tumor microenvironment for distinct lymphocyte subpopulations was determined by immunohistochemistry. (A) Cryosections of primary tumors induced with NXS2 wild type, NXS2 mock transfected and NXS2-FKN cells were stained with mAbs specific for CD45 (pan leukocyte marker), CD4 and CD8 (T-cell subpopulations). Each panel shows photographs taken at 400x of representative areas within distinct primary tumors. Black arrows indicate infiltrating cells with characterisitc red membrane staining. (B) The number of tumor infiltrating cells was quantified by counting the total number of infiltrating cells per high power field (HPF) at 400x. Bars represent MV ± SD of ten HPFs. The differences between mice receiving NXS2-FKN cells and all control groups were statistically significant (*p<0.01).

Fig. 7 Effect of FKN immunogenetherapy combined with targeted IL-2 on primary tumor growth. The anti-tumor immune response combining FKN immunogenetherapy with targeted IL-2 was determined following the experimental design as depicted in Figure 2. Experimental groups of mice (n=6) received s.c. injections with 2x10⁶ NXS2-FKN cells and NXS2 mock transfected cells. 5 days after s.c. injection, mice (n=6) were treated with 5 daily injections of tumor specific anti-ganglioside GD2 antibody ch14.18-IL-2 fusion protein (5x5 µg). Mice (n=6) treated with a non-specific anti-human EGF receptor antibody ch225-IL-2 fusion protein (5x5 µg) were used as a control group. (A) Primary tumor growth was monitored over time by microcaliper measurements and the tumor size was calculated according to ½ x width² x length. Data points represent MV ± SD. (B) The primary tumor weight was determined following surgical removal 17 days after s.c. tumor inoculation. Bars represent MV ± SD. The difference between the FKN immunogenetherapy and targeted IL-2 combination group and mock control group was statistically significant (*p<0.01).

Fig. 8 Effect of FKN immunogenetherapy combined with targeted IL-2 on experimental liver metastasis. The anti-tumor immune response combining FKN immunogenetherapy with targeted IL-2 was determined following the experimental design as depicted in Figure 2 using the same experimental groups as described for results shown in Fig.7. All mice (n=6) received a lethal intravenous challenge with 10⁵ NXS2 wild type cells one day after removal of the primary tumor. The level of experimental liver metastasis was determined 3 weeks after i.v. challenge. (A) Liver metastases were scored according to the coverage of the liver surface with neuroblastoma metastases as follows: 0% = 0, <20% = 1, 20 – 50% = 2, >50% = 3. (B) The level of liver metastasis was assessed by a determination of the wet liver weight. Bars represent MV ± SD, n=6. The difference between the FKN immunogenetherapy and targeted IL-2 combination group and all control groups was statistically significant (*p<0.05).

Fig. 9 Determination of the CTL response induced by FKN immunogenetherapy combined with targeted IL-2. The CTL response in mice receiving FKN immunogenetherapy combined with targeted IL-2 was determined by a standard ⁵¹Cr release assay at varying effector to target cell (E/T) ratios. For this purpose, pooled splenocytes of all experimental groups of mice (n=6) were harvested at the end of the in vivo experiment (Fig. 2), and used after a 4-day in vitro stimulation phase as described in material and methods. CTL activity was determined in the absence (triangle) and presence (square) of anti-MHC class I antibody (anti-H-2K^k, 25µg/ml). Results show cytotoxicity in percent (MV ± SD) of experiments in triplicate.

Fig. 10 Analysis of the T cell activation following FKN immunogenetherapy combined with targeted IL-2. T-cell activation was determined in pooled splenocytes of experimental groups of mice (n=6) at the end of the in vivo experiment (Fig. 2), after the 4 day in vitro stimulation phase as described in material and methods. (A) The secretion of IFN-γ and TNF-α of distinct T cell subpopulations was determined by two-color flow cytometry. (B) T cell activation markers CD25 and CD69 of distinct T cell subpopulations were determined by two-color flow cytometry. Data represent the relative increase over naïve control splenocytes in percent (MV ± SD) of experiments in triplicate. The difference between the FKN immunogenetherapy and targeted IL-2 combination group and mock control group was statistically significant (*p<0.01).

Fig. 11 Analysis of the number of CD4 ⁺ and CD8 ⁺ T-cells following FKN immunogenetherapy combined with targeted IL-2. The number of CD4⁺ and CD8⁺ T-cells was determined by two-color flow cytometry in pooled splenocytes of experimental groups of mice (n=6) at the end of the in vivo experiment (Fig. 2), prior to the 4-day in vitro stimulation phase. Data represent the relative increase over naïve control splenocytes in per cent (MV ± SD) of experiments in triplicate. The difference between the FKN immunogenetherapy and targeted IL-2 combination group and mock control group was statistically significant (*p<0.01).

Fig. 12 Effect of T cell depletion on FKN immunogenetherapy combined with targeted IL-2 on primary tumor growth. The role of CD4⁺ or CD8⁺ T cells in mediating the anti-tumor immune response following a combination of FKN immunogenetherapy with targeted IL-2 was determined using the experimental design as depicted in Figure 2. Experimental groups of mice (n=6) received s.c. injections with 2x10⁶ NXS2-FKN cells and NXS2 mock transfected cells. 5 days after s.c. injection, mice (n=6) were treated with 5 daily injections of tumor specific anti-ganglioside GD2 antibody ch14.18-IL-2 fusion protein (5x5 µg). Mice (n=6) were depleted of CD4⁺ or CD8⁺ T cells by intraperitoneal injection of 200 µg of anti-CD4 mAb (RM4-5) or anti-CD8 mAb (53-6.7) on days –1, 7 and 14. The efficacy of this depletion was previously described (20). Primary tumor growth was monitored over time by microcaliper measurements and the tumor size was calculated according to (½ x width² x length). Data points represent MV ± SD. The difference between the non-depleted group and all control groups was statistically significant (*p<0.05).