3 Results

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The following chapters summarize all the results obtained with this new immunotherapy concept. Novel, yet unpublished data are described in more detail (Fig. 3-12). The experimental design used throughout the manuscript is depicted in figure 2.

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

3.1 Construction of a mammalian expression vector encoding mFKN

The gene encoding for murine FKN was successfully cloned from the murine breast cancer cell line D2F2 by RT-PCR and subcloned into the mammalian expression vector pIRES, namely pIRES-FKN, using NheI and EcoRI restriction enzymes [4]. The mFKN sequence was verified by molecular sequencing.

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

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The successful transfection of pIRES-FKN into NXS2 cells and stable transcription of FKN in NXS2-FKN tumor tissue were confirmed by RT-PCR. The amplification of FKN cDNA (1118bp) was only found in NXS2-FKN cells in contrast to NXS2 parental and mock transfected controls (Fig. 3). Similar results were obtained from tumor tissue with NXS2-FKN cells in vivo [4]. GAPDH was used as a housekeeping gene (300 bp) to verify the integrity of RNA and cDNA preparations.

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.

The expression of the FKN protein in the secreted and membrane bound form was assessed by ELISA (Fig. 4A) and FACS (Fig. 4B), respectively. In FKN-NXS2 bulk culture cells, the level of soluble form of FKN was quantified at a rate of 5.5±0.48 ng/ml/24hand flow cytometry demonstrated 31.32% of these cells express membrane bound form of FKN. After 2 rounds of subcloning, a NXS2-FKN subclone was obtained that showed a higher secretion rate of 14±1.04 ng/ml/24hand 75.32% cells expressed FKN bound to the cell surface. Furthermore, the continuous expression of the FKN protein was also determined by immunohistochemistry in NXS2-FKN tumor tissue in contrast to parental NXS2 and NXS2 mock-transfected controls [4].These findings demonstrate efficient transcription and expression of FKN from my DNA construct in vitro and in vivo.

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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.

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

Chemotaxis mediated by supernatants from NXS2-FKN cells was tested in a boyden chamber assay in vitro, which revealed a maximum of 30% of migrated cells at a concentration of 47.6ng/ml (Fig. 5). This finding was in contrast to the negative control. This chemotactic activity can be partially blocked by adding anti-murine FKN Abs (2 µg/ml) to supernatants suggesting that it was specifically mediated by FKN in this assay. Recombinant FKN from R&D company served as a positive control. Interestingly, migration included recombinant FKN followed a ‘bell-shaped’ characteristics with a decrease at higher concentration, a phenomenon known, but not well understood in chemotaxis assay.

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).

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In order to determine FKN mediated chemotaxis in vivo, the migration of leukocytes into primary tumors was analyzed by immunohistochemistry. Primary tumors were induced by s.c. injection of 2 x 106 NXS2-FKN, NXS2-mock, NXS2 parental cells. Immunohistochemistry was performed 16 days after tumor cellinoculation. Effective migration of leukocytes into FKN producing primary tumors was clearly demonstrated, indicated by a factor 3 increase over the NXS2 wild type and mock transfected controls (Fig. 6A 6B). Interestingly, the highest increase in migration was observed in the CD8+ T-cell subpopulation (factor 7).

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).

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

The effect of targeted IL-2 on FKN gene therapy was first evaluated on primary tumor growth as previously described [4]. Transfection of NXS2 cells with FKN induced a limited decrease of tumor growth rate (Fig. 7A) and tumor weight (Fig. 7B). This finding was in contrast to the simultaneous administration of ch14.18-IL-2, which resulted in a marked inhibition of primary tumor growth. Importantly, one third of mice showed a complete tumor rejection only in the FKN/ch14.18-IL-2 combination group. The effect of ch14.18-IL-2 was specific since a non-specific control using ch225-IL-2 had no additional effect on FKN gene therapy as indicated by an average tumor size of 297.7 ± 35.1 mm3, which is not different from the average tumor size of FKN monotherapy at 315.3 ± 67.3 mm3 (p > 0.1).

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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).

The efficacy of this combination therapy against liver metastasis was determined following a lethal challenge with 105 wild type NXS2 cells i.v. Importantly, 5/6 mice challenged with NXS2 wild type cells receiving the FKN and ch14.18-IL-2 combination therapy were free of the experimental liver metastasis (Fig. 8A) and the livers of mice in this group revealed a normal weight (Fig. 8B), which is around 1 gram. This finding was in contrast to the livers from naive control and unspecific control groups.

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).

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

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Splenocytes receiving FKN and ch14.18-IL-2 revealed a 3-fold increase of the cytolytic response against NXS2 cells in contrast to the mock control group. Importantly, no lysis was observed against NXS2 cells in splenocytes from mice treated with NXS2-FKN alone or combined with ch225-IL-2 (Fig. 9). The lysis observed in the NXS2-FKN plus ch14.18-IL-2 group can be inhibited by adding anti-MHC-І (H-2Kk) antibody, indicating MHC class І restriction, characteristic for a CD8+ T-cell response. These findings clearly correlate with in vivo findings and demonstrate the induction of a CTL response only following this combination treatment regimen. This contention is supported by the fact that neither NXS2-FKN nor ch14.18-IL-2 used as monotherapy could achieve such a CTL response.

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-2Kk, 25µg/ml). Results show cytotoxicity in percent (MV ± SD) of experiments in triplicate.

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

In order to determine the level of T cell activation, the expression of T cell activation markers and the production of pro-inflammatory cytokines by distinct T cell populations were tested by flow cytometry.

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Interestingly, it was observed that the highest increase of T cell activation markers (CD69, CD25) (Fig. 10A) and proinflammatory cytokines (TNF-α, INF-γ)#SYMBOL#(Fig. 10B) were in the NXS2-FKN and ch14.18-IL-2 combination group over all control groups including the NXS2-FKN group receiving non-specific ch225-IL-2 fusion protein. This finding clearly demonstrated superior efficacy and specificity of combining targeted IL-2 with FKN gene therapy in inducing activated TH1 CD8+-T-cells.

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).

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

In order to further elucidate a role for T-cells in this treatment, the number of CD8+ and CD4+ T cells was determined in splenocytes harvested from treated animals by flow cytometry (Fig. 11). The number of CD3+/CD4+ T cells rose 1.83 times in mice receiving FKN gene therapy and 2.46 times in the FKN gene therapy and ch14.18-IL2 combination group over the mock control group. Similarly, the number of CD8+/CD3+ T cells was markedly increased in the combination therapy group, but only slightly increased in FKN gene therapy used as monotherapy or FKN gene therapy combined with ch225-IL-2 unspecific control. This result suggested that CD4+ and CD8+ T cells proliferated to the highest extent in the FKN genetherapy group combined with targeted IL-2.

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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).

Depletion assay further supported this conclusion (Fig. 12). The depletions of CD4+ T cells and CD8+ T cells were accomplished by injection of anti-CD4- and anti-CD8-antibody in vivo. As shown in Figure 12, FKN gene therapy and ch14.18-IL-2 induced significant inhibition of tumor growth in non-depleted mice. In contrast, the primary tumor growth rate increased in CD4- or CD8-depleted mice, indicating that the antitumor effect induced by NXS2-FKN and targeted IL-2 was mediated by both CD4+ T cells and CD8+ T cells.

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).


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