IJC International Journal of Cancer
Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNc treatment in mouse tumor models Filip Van Hauwermeiren1,2, Roosmarijn E. Vandenbroucke1,2, Lynda Grine1,2, Leen Puime`ge1,2, Elien Van Wonterghem1,2, Hong Zhang3 and Claude Libert1,2 1
Inflammation Research Center (IRC), VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 3 Isis Pharmaceuticals, Carlsbad, CA 2
Cancer Therapy
Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55-deficient mice. In previous work we demonstrated that P551/2 mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNc) remain fully functional in these mice. Hence, a high dose of TNF/IFNc has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNc treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNc antitumor therapy.
Tumor necrosis factor (TNF) is a pleiotropic cytokine with an important role in diverse cellular processes, such as proliferation, differentiation, cell death and cell survival. It is best known for its key role in many inflammatory and autoimmune diseases, such as Crohn’s disease and rheumatoid arthritis.1 However, TNF was originally identified and named after its potent antitumor effects, especially in combination with interferon gamma (IFNg) and/or chemotherapeutics.2 Unfortunately, owing to its strong proinflammatory actions, its injection also causes systemic inflammation that can lead to life-threatening shock.3 Because the anticancer therapeutic Key words: TNF/IFNg therapy, TNFR1, antisense oligonucleotides, toxicity Additional Supporting Information may be found in the online version of this article Conflict of interest: H.Z. was an employee of Isis Pharmaceuticals Grant sponsors: Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), Vlaamse Liga tegen kanker (VLK), Stichting tegen de Kanker, BOF-UGent, the Research Foundation Flanders (FWO Vlaanderen), the Interuniversity Attraction Poles Program of the Belgian Science Policy DOI: 10.1002/ijc.28704 History: Received 11 Oct 2013; Accepted 11 Dec 2013; Online 31 Dec 2013 Correspondence to: Prof. Claude Libert, VIB IRC UGENT, Technologiepark 927, 9000 Ghent, Belgium, Fax: 1003292217673, E-mail: [email protected]
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dose of TNF is close to the maximally tolerated dose (MTD), its current use in anticancer therapy is limited to local settings in which systemic TNF actions are prevented, such as in isolated limb perfusion. This locoregional administration of TNF is highly effective, induces limited toxicity and is used as limb-sparing treatment for in transit melanoma and soft tissue sarcoma.4,5 The antitumor effect of TNF/IFNg therapy is mediated by inhibition of avb3 integrin, which is specifically expressed by neovascular endothelium cells and allows them to attach to the extracellular matrix.6 Loss of adhesion of these endothelial cells causes cell death, increased vascular permeability and eventually complete destruction of the tumor neovasculature. Interestingly, TNF-induced inflammation and toxicity is caused by a signaling cascade that culminates in activation of MAPKs and transcription factors such as NFjB,7 leading to the induction of multiple toxic mediators and ultimately to TNF-induced lethal inflammation.8 Therefore, separating those two biological activities seemed possible and several strategies have been proposed by different research groups to limit the toxicity of TNF and to increase its therapeutic potential in antitumor treatment. Multiple TNF mutants or TNF fusion proteins have been produced to limit toxicity or increase TNF accumulation in tumor tissues, e.g., TNFerade (a radiation-inducible TNF gene),9 chemically modified TNF, TNF mutants with lower toxicity10–12 and TNF fusion proteins (e.g., NGR-TNF) to target TNF to the tumor neovasculature.13
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What’s new? Tumor necrosis factor (TNF) can produce potent antitumor effects as well as potentially life-threatening systemic inflammation, which has limited its therapeutic use. Different strategies to overcome systemic toxicity have been explored, including the inhibition of mediators of TNF-induced toxicity. Here, antisense oligonucleotides targeted against TNFR1 (P55), the primary mediating receptor in models of both acute TNF toxicity and TNF/IFNc antitumor activity, were found to effectively lower TNFR1 protein levels in select tissues and thereby reduce toxicity associated with TNF/IFNc therapy, without affecting antitumor activity. The findings provide new evidence for the applicability and safety of TNF/IFNc therapy.
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targeting SOD1,24 elevated plasma LDL levels by targeting apoB-10025 and cancer by targeting multiple oncogenes.26 In our study, treatment with P55-targeting ASOs resulted in a strong downregulation of P55 protein levels in liver, blood mononuclear cells and small intestine. This effect was sufficient to protect mice against TNF toxicity in both acute and antitumor settings in several tumor models.
Material and Methods Mice
Female C57BL6, NMRI Nu/Nu and B6CBAF1 mice 8–12 weeks of age (Janvier, France) were maintained in conventional, temperature-controlled, air-conditioned animal houses with 14–10 hr light/dark cycle, and received food and water ad libitum. All experiments were approved by the local ethics committee. Oligonucleotide treatments
Mice were injected i.p. every other day for a total of four times with 25 mg/kg 20 -O-(2-methoxy)ethyl-modified ASO specific for P55 (ISIS 108426) or nonspecific control (ISIS 141923) dissolved in 200 ml phosphate-buffered saline (PBS). For the ASO safety studies in an antitumor setting, ASOs were injected i.p. at 25 mg/kg on days –7, –5, –3, –1, 1, 3, 5, 7 and 9 relative to the start of TNF/IFNg treatment. The ASOs were a kind gift from Isis Pharmaceuticals. P55 ELISA
Whole organ samples were isolated for kidney, spleen, liver and lung. We isolated intestinal epithelial cells by scraping them from different parts of the small intestine. Mononuclear cells were isolated after separation of 300 ml blood on a Ficoll gradient. Samples were homogenized in ice-cold buffer [PBS, 0.5% CHAPS and protease inhibitors (Complete, Roche, Vilvoorde, Belgium)]. Homogenates were centrifuged for 30 min at 20,000g and 4 C and the supernatant was stored at –80 C. Protein concentration was determined by the Bradford method (BioRad, Eke, Belgium). P55 levels were determined with the Quantikine sP55 ELISA kit (R&D Abingdon, UK). The levels were normalized to the P551/1 levels of untreated samples, which were set as 100%. Cytokines
Recombinant mouse TNF and IFNg were expressed in Escherichia coli and purified in our laboratory. TNF had a specific
Cancer Therapy
Another possibility is to focus on the identification and inhibition of TNF-induced toxic mediators while maintaining TNF’s antitumor potential. Previously, we identified multiple TNF-induced toxic mediators, including several members of the family of matrix metalloproteinases,14 IL17,15 necroptosis16 and type I interferons.17 More recently, we identified P55 (TNFR1 and CD120a) as a promising target for overcoming the toxicity of systemic TNF antitumor treatment.8 TNF acts by binding two different receptors, P55 and P75 (TNFR2 and CD120b).18 P55 has important physiological functions, possesses anticancer activity and contributes to pathological inflammatory processes,19 whereas P75 has been implicated mainly in immune modulation.1 P55 is the main receptor involved in both the acute TNF toxicity model and the TNF/IFNg antitumor model. TNF-induced tumor regression and toxicity are absent in P55-deficient mice, whereas P75-deficient mice behave like wild-type mice in these models.20 In previous work we found that P551/2 mice have solid protection against TNF toxicity. In TNF/IFNg antitumor treatment, P551/2 mice resist TNF toxicity of doses up to tenfold the LD50 for P551/1 mice, but the antitumor effects remain intact. In response to TNF, P551/2 mice have a strongly reduced induction of inflammatory signaling pathways and much reduced production of several toxic mediators.8 Moreover, although we reported before that the liver is an essential target of TNF’s lethal actions,14 we recently identified P55 expression levels on intestinal epithelial cells (IECs) as crucial determinants of systemic TNF toxicity. P551/1 expression specifically in the IEC is sufficient to induce TNF toxicity, while P551/2 or P552/2 expression in IEC is protective against TNF toxicity.8 Here, we wanted to mimic the phenotype of P551/2 mice by downregulating P55 expression levels using antisense oligonucleotides (ASOs, Isis Pharmaceuticals, Carlsbad, CA). These ASOs are 20 -O-(2-methoxyethyl) chemically stabilized oligonucleotides which are nuclease resistant and show increased affinity for their target sequence compared to unmodified oligonucleotides.21 They bind to their target mRNA and modify its function by different mechanisms, e.g., inducing its degradation, causing exon skipping or blocking translation.22 ASO-based therapies are now in clinical trials for various pathologies and show promising results also for the treatment of Duchenne muscular dystrophy (DMD) by targeting dystrophin,23 amyotrophic lateral sclerosis (ALS) by
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activity of 1.0 3 109 IU/mg. IFNg had a specific activity of 1.1 3 109 IU/mg. Endotoxin in the cytokine preparations was below the detection limit of a Limulus amoebocyte lysate assay. Cytokines and LPS were diluted in endotoxin-free PBS (GIBCO, Invitrogen, Merelbeke, Belgium) immediately before injection.
percent tumor area was determined by photographing the surface of each lung lobe’s front and back. As these tumors were black, it was possible to measure their areas with ImageJ software. Percent tumor area was calculated as the number of “black tumor” pixels divided by the total number of pixels in the lung area.
Acute TNF-bolus model
Statistical analysis
Mice were injected i.p. with mouse TNF diluted in 200 ml of PBS. Lethality was monitored for 72 hr, after which no further deaths occurred and body temperatures of surviving mice returned to normal.
Survival curves (Kaplan–Meier plots) were compared using a log-rank test. Means 6 SD were compared with a Student’s t-test. Final lethality data were compared using a Fisher’s exact test. *, ** and *** represent 0.01 < p < 0.05, 0.001 < p < 0.01 and p < 0.001, respectively.
Temperature measurement and blood collection
Rectal body temperature was measured with a digital thermometer. Blood was taken with a glass capillary from the tail vein and allowed to clot for 30 min at 37 C. The clot was removed and serum was isolated by centrifugation at 20,000g for 10 min and stored at 220 C. NOx determination in serum samples
NOx concentration in the serum (sum of the stable NO metabolites nitrate and nitrite) was determined as described.27 Determination of IL-6 in serum
IL-6 was measured as described before, i.e., by using an IL-6dependent 7TD1 hybridoma cell line.28
Cancer Therapy
Tumor cell cultures and antitumor experiments Subcutaneous B16BL6 melanoma. The murine B16BL6
melanoma cell line was a gift from M. Mareel (Ghent, Belgium) by courtesy of I. Fidler (Dallas, USA). Cultured B16BL6 cells were washed thrice in PBS and counted. Their density was adjusted with PBS and 100 ll containing 6 3 105 cells was injected subcutaneously (s.c.) in the shaved right thigh of mice. Treatment was started 12 days later by s.c. injection with 0.1 ml of a mixture of TNF and IFNg next to the tumor. This treatment was repeated daily for 10 days. Lethality and tumor size index (TSI) were scored. TSI was defined as the product of longest diameter of the tumor and the diameter perpendicular to it (mm2). Subcutaneous Lewis lung carcinoma. LLC cells were washed
thrice with PBS and counted. Their density was adjusted with PBS and 100 ml containing 6 3 106 cells was injected s.c. in the shaved right thigh of mice. Treatment was the same as for the B16BL6 melanoma model. Lung colony model. B16BL6 melanoma cells were washed
thrice in PBS and counted. Their density was adjusted with PBS and 200 ml containing 5 3 104 cells was injected in the tail vein of the mice. Treatment was started 15 days later by i.p. injection of 0.3 ml of PBS containing 10 or 50 mg TNF. This treatment was repeated daily for 10 days. Lethality was scored during this period and at the end of the treatment the
Results ASO treatment reduces P55 protein levels in liver and small intestine
We first investigated which organs are affected by the P55 ASO treatment. We based our protocol on a published injection schedule that resulted in 50% downregulation of P55 mRNA expression in the liver.29 Mice were injected intraperitoneally (i.p.) with control (CTR) ASO or P55 ASO treatment (25 mg/kg) every other day, four times in total (Fig. 1a). Twenty-four hours after the fourth ASO treatment, mice were sacrificed, tissues were isolated and processed and P55 protein levels were measured by ELISA. Treatment with P55 ASO caused a reduction in P55 protein levels in the liver (50%), blood mononuclear cells (75%) and different parts of the small intestine (75%), but not in lung, spleen and kidney (Fig. 1b). ASO-mediated P55 downregulation protects against acute TNF toxicity
We studied the effect of P55 downregulation in the acute TNF bolus model. Mice were treated four times with control ASO or P55 ASO (25 mg/kg) every other day, and 24 hr after the fourth treatment they were injected i.p. with 25 mg TNF, the LD100 for untreated animals (Fig. 2a). TNF injection is known to cause acute and lethal inflammatory shock associated with hypothermia, hypotension and abundant release of cytokines.8 Mice pretreated with P55 ASOs experienced milder hypothermia and were significantly protected against death (30% lethality) compared to CTR ASO-treated mice (80% lethality) (Figs. 2b and 2c). In a separate experiment, blood was collected from mice before and 3 hr after TNF injection, serum was prepared and TNF-induced NO metabolites and IL6 levels were measured. Mice treated with P55 ASO had significantly lower levels of NO metabolites (Fig. 2d) and serum IL6 (Fig. 2e) compared with CTR ASOtreated mice. P55 ASO treatment protects against TNF/IFNc toxicity in the B16Bl6 melanoma tumor model
In contrast to the acute TNF model, which is based on injection of a single bolus, our established and validated TNF/ C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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IFNg antitumor model requires daily TNF/IFNg injections for 10 days to induce complete tumor regression.2,8 We first used the B16BL6 melanoma model in which tumor cells are injected in the right thigh of mice and left to grow for 10 days to a size of about 1 cm2. Mice were injected i.p. every other day nine times with control ASO or P55 ASO (25 mg/ kg), four times during tumor growth and five times during TNF/IFNg treatment (Fig. 3a). No significant effects on tumor growth were observed when comparing P55 ASO with CTR ASO or PBS treatment (data not shown). Before the start of the actual TNF/IFNg treatment, P55 protein levels were measured in the liver after each ASO injection to confirm that the effect of ASOs on P55 was not influenced by growing tumors. Similar to the four times P55 ASO treatment described above, a stable downregulation of about 50% was observed in the livers of mice treated with P55 ASO throughout the whole period of tumor growth. In contrast, CTR ASO treatment had no effect on P55 levels. P55 levels in the inoculated tumors were also measured during tumor growth, but they were not affected by P55 ASO treatment, which is appropriate for a full P55-mediated antitumor response (Figs. 3b and 3c). In the next experiment, mice pretreated with ASOs were injected daily with PBS or 17.5 mg TNF and 5,000 IU IFNg C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
for 10 days and tumor size and lethality were monitored daily. After 10 days of TNF/IFNg treatment, death of mice was monitored for 10 days and then the survivors were euthanized. CTR ASO or P55 ASO treatment on its own did not influence tumor growth in the PBS-treated groups. Daily TNF/IFNg treatment caused strong tumor regression in mice cotreated with CTR ASOs or P55 ASOs (Fig. 3d). However, P55 ASO cotreated mice were significantly protected against the toxic effects and survived the antitumor treatment, in contrast to CTR ASO cotreated animals, all of which died from TNF toxicity during the study (Fig. 3e). Similar results using the B16Bl6 model were obtained in genetic backgrounds other than C57BL/6, namely NMRI Nu/Nu and B6CBAF1 mice (Supporting Information Figs. 1A–1D). P55 ASO treatment protects against TNF/IFNc toxicity in other tumor models
To confirm the therapeutic value of P55 ASO cotreatment, two other cancer models were used. In both models TNF/ IFNg and ASO cotreatment was identical to the treatment schedule used for the B16Bl6 tumor model (Fig. 3a). One model was the Lewis lung carcinoma (LLC) tumor model.30 LLC cells were injected in the right thigh of mice and the tumors were allowed to grow to about 1 cm2. Tumor-bearing
Cancer Therapy
Figure 1. Effects of P55 ASO treatment on P55 protein levels in multiple organs. (a) ASO pretreatment schedule. Mice were pretreated four times with 25 mg/kg CTR or P55 ASO. Twenty-four hours after the last pretreatment mice were sacrificed and organs were collected. (b) P55 ELISA after treatment with P55ASO. Mice were treated with P55 ASO (n 5 5) or control ASO (n 5 5). P55 ASO resulted in significant downregulation of P55 in the liver, small intestine and blood mononuclear cells compared to CTR ASO, but no difference was observed in P55 levels in lung, spleen and kidney.
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Cancer Therapy
Figure 2. P55 ASO treatment protects against acute TNF toxicity. (a) ASO pretreatment schedule. Mice were pretreated four times with 25 mg/kg CTR or P55 ASO. Twenty-four hours after the last pretreatment mice were injected i.p. with 30 mg TNF. (b and c) Survival after acute TNF treatment. Mice treated i.p. with P55 ASO (green, •, n 5 19) were significantly protected against a single injection of TNF compared to control ASO-treated mice (red, 䊏, n 5 15) and experienced milder hypothermia (c). (d and e) NOx levels and IL6 levels 3 hr after i.p. injection of 30 mg TNF in P55 ASO-treated mice. Induction of IL6 and NOx was weaker in P55 ASO-treated mice (n 5 10) than in mice treated with control ASO (n 5 9).
mice were then pretreated with CTR or P55 ASOs as described for the B16Bl6 model, and the tumors were injected paralesionally with 17.5 mg TNF and 5,000 IU IFNg. Again, P55 ASO cotreatment allowed full antitumor effects and resulted in significantly reduced toxicity (only 10% lethality in contrast to 90% in CTR ASO-treated mice) (Figs. 4a and 4b). We also used the B16Bl6 lung colony model. Intravenous injection of 5 3 104 melanoma cells resulted in the formation of lung nodules, which were allowed to grow for 15 days. Then, mice were treated i.p. with 15 mg TNF and 5,000 IU IFNg daily for 10 days. Only mice surviving the 10-day treatment were considered for tumor growth evaluation. Tumor burden was analyzed by calculating the percentage of black melanoma surface area on the individual lung lobes. Compared to the PBS-treated groups, the TNF/IFNg-treated groups showed reduced tumor burden: 10% reduction for the CTR ASO cotreated mice and 18% reduction for the P55 ASO cotreated mice. P55 ASO cotreated mice were protected
against TNF/IFNg treatment and all of them survived the treatment, whereas only 45% of CTR ASO cotreated mice survived (Figs. 4c and 4d). Calculation of the maximum tolerable dose in P55 ASO-treated mice
Finally, we determined the toxicity profile of daily TNF/IFNg therapy for 10 days in B16Bl6 tumor-bearing mice cotreated with control ASOs or P55 ASOs according to the same injection schedule (Fig. 3a). Tumors were inoculated and left to grow for 10 days, after which mice were injected daily with TNF (doses ranging from 10 to 25 mg per day per mouse), combined with 5,000 IU IFNg. Death of mice was followed during the 10 days of therapy with TNF/IFNg and during a follow-up period of 10 days. After that the remaining mice were euthanized. Treatment with CTR ASO resulted in an LD50 of 15 mg TNF, while P55 ASO-treated mice reached a 50% higher LD50 of 22.5 mg TNF (Figs. 5a and 5b and Supporting Information Fig. 2). C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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Discussion TNF possesses potent antitumor properties but their full exploitation is hampered by its strong proinflammatory effects. Nevertheless, the clinical use of TNF, along with studies in mice, supports the view that TNF has spectacular anticancer effects on a wide variety of solid tumors.2,5,31 We recently identified P55 as an excellent therapeutic target to reduce the toxicity of systemic TNF/IFNg antitumor therapy.8 P551/2 mice are solidly protected against TNF toxicity in TNF/IFNg antitumor models, while retaining the potent TNF-induced antitumor effects. To provide proof of principle for pharmacologic treatment, we used P55 targeting ASOs to mimic the P551/2 situation. We made use of the second-generation ASOs, 20 -Omethoxyethyl (20 MOE)-modified ASOs (Isis Pharmaceuticals), which have increased stability owing to their RNase resistance C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
and increased affinity for target mRNA compared to the firstgeneration phosphorothioate oligonucleotides.32 ASOs can strongly reduce protein levels of a target gene, e.g., by recognition of the mRNA–ASO complex, resembling a DNA–RNA intermediate, which gets selectively degraded by RNase H.33 However, the effectiveness of therapy largely depends on delivery to the desired target organ or cell type. Pharmacokinetic studies indicate that systemic delivery of naked ASOs leads mainly to liver and kidney targeting whereas spleen and intestine are targeted with lower efficacy.34,35 In our study, we observed that systemic treatment with P55 ASO reduces P55 protein levels in liver, blood mononuclear cells and IECs but not in lung, kidney, spleen or tumor tissue. Interestingly, treatment with P55-specific ASOs protected against acute TNF toxicity. Upon TNF injection, mice treated with P55 ASO had significantly better survival, a
Cancer Therapy
Figure 3. P55 ASO treatment protects against TNF toxicity in TNF/IFNg antitumor treatment. (a) ASO pretreatment and TNF/IFNg injection schedule. Mice were inoculated with 6 3 105 B16Bl6 cells on day –10 and treated nine times with 25 mg/kg CTR or P55 ASO starting on day –9. Daily treatment with TNF/IFNg started on day 0. (b and c) P55 ELISA after treatment of tumor-bearing mice with ASO but without TNF/IFNg treatment. Liver P55 levels in P55 ASO-treated mice (n 5 4 per time point) were significantly reduced compared to control ASOtreated mice. No significant downregulation of P55 was found in tumor tissue after treatment with P55 ASO. (d and e) Antitumor effects and survival of ASO-treated mice in an antitumor experiment. The P55 ASO-treated mice (•, n 5 10) had better survival than control ASO mice (䊏, n 5 10) when treated daily with 17.5 mg TNF and 5,000 IU IFNg. No significant differences between the groups were found in tumor regression and tumor growth (empty symbols stand for PBS and filled symbols stand for TNF/IFNg treatment).
Cancer Therapy
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Figure 4. P55 ASO treatment protects against TNF/IFNg toxicity in LLC and B16Bl6 lung colony model. (a and b) Antitumor effects (a) and survival (b) of ASO-treated mice in a LLC antitumor experiment. Mice were first inoculated s.c. with 6 3 106 LLC cells and then treated with ASOs as described for the B16BL6 model above. Finally, they were treated for 10 days with 17.5 mg TNF 1 5,000 IU IFNg or with PBS. Tumors regressed well in all groups but only the P55 ASO-treated mice (green, • n 5 9) had significantly better survival than the control ASO-treated mice (red, 䊏, n 5 9). (c and d) Antitumor effects and survival of ASO-treated mice in the B16Bl6 lung colony model. Mice were injected i.v. with 50,000 B16Bl6 melanoma cells, and after 15 days TNF/IFNg or PBS treatment was initiated. There was a small reduction of tumor area in all TNF/IFNg-treated groups but P55 ASO (n 5 8)-treated mice had the better survival compared to CTR (n 5 9).
milder drop in body temperature and weaker induction of the inflammatory mediators IL6 and NO. This protection was also observed in the TNF/IFNg antitumor model: P55 ASO-treated mice had the best outcome, with remarkable tumor regression and significantly better survival than the PBS- or CTR ASO-treated mice. Such tumor regression without the associated TNF toxicity was also obtained in different tumor models as well as in different genetic backgrounds, indicating that it might be a widely applicable therapeutic option. Finally, P55 ASO therapy increased the maximum tolerated TNF dose (MTD) by 50% compared to CTR ASOtreated mice. These data provide proof of principle that P55
ASO treatment reduces P55 expression and increases the therapeutic potential of TNF in cancer therapy. Although MTD increased 50% by P55 ASO therapy, we could not mimic the P551/2 phenotype completely, which had a tenfold higher MTD.8 This is likely due to nonsystemic P55 downregulation in a rather limited set of organs (liver, IECs and blood mononuclear cells). Better results can be expected when P55 protein levels are also downregulated in other cell types that mediate TNF toxicity. To increase the uptake in target tissues different strategies can be used. Modifications can be used to optimize the delivery of ASOs to the target tissues: liposome encapsulation, cholesterol modification C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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Figure 5. P55 ASO pretreatment increases the maximum tolerable dose of TNF in a TNF/IFNg antitumor treatment. (a) Lethality data from the toxicity study of TNF/IFNg-treated B16BL6-inoculated mice treated with CTR ASOs or P55 ASOs. (b) Summary of the lethality data. LD50 was 15 mg in CTR ASO-treated mice and 22.5 mg in P55 ASO-treated mice.
can protect mice in a model of colitis.37 P55 inhibition might be an interesting alternative to current anti-TNF therapies. By specifically inhibiting P55 signaling instead of completely blocking TNF, the often beneficial and immune modulatory P75 signaling is kept intact, and this might enhance repair functions and reduce anti-TNF-associated side effects, as observed, e.g., in multiple sclerosis.1,38,39 In summary, we demonstrate that reduction of P55 expression by P55 ASO increases the safety of TNF in anticancer therapy without loss of anticancer efficacy. There is still room for improvement by using better delivery systems for broader downregulation of P55 protein expression in other tissues that mediate TNF toxicity. Identification of these tissues and development of strategies to target them will further increase the MTD of TNF and might allow broader applicability and systemic exploitation of the remarkable antitumor effects of TNF therapy.
Acknowledgements This work was supported by grants to CL from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWTVlaanderen), Vlaamse Liga tegen kanker (VLK), Stichting tegen de Kanker, BOF-UGent, the Research Foundation Flanders (FWO Vlaanderen) and the Interuniversity Attraction Poles Program of the Belgian Science Policy. The authors wish to thank Dr. Amin Bredan for editing the manuscript. They also thank Isis Pharmaceuticals for providing antisense oligos and Wilma Burm and Joke Vanden Berghe for excellent technical assistance. CL and FVH formulated the hypothesis and initiated and organized the study. FVH, REV, LP, LG and EVW performed the experimental work and analyzed the data. CL oversaw the experiments and data. HZ and CL provided essential materials and funding. FVH drafted the manuscript and CL finalized it.
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Cancer Therapy
or conjugation to receptor-specific ligands.36 In addition, clinicians are studying many routes of delivery, including aerosol, enema, intrathecal, intravenous, subcutaneous, topical and intravitreal to further optimize uptake of ASO in the desired tissues. Recently, we identified P55 expression levels in IECs as an important determinant of TNF toxicity.8 Our experiments on mice with conditional, Villin-cre-induced P55 reactivation showed that P551/1 expression in IECs is sufficient to cause TNF toxicity, while experiments on mice conditionally deficient in P55 indicated that a P551/2 or P552/2 status in the IECs is protective. However, like the P55 ASO-induced protection, conditional deletion in IECs does not provide the level of protection observed in P551/2 mice, confirming that other cell types than IECs are involved in mediating TNF toxicity. Similar experiments performed in liver (albumin Cre)- and myeloid (LysM Cre)-specific P55 conditional reactivation and deficiency uncovered only a minor role for liver or myeloid cells in TNF toxicity. To assess the contribution of specific organs to TNF toxicity, we are extending these studies with additional cre-mediated organ-specific (e.g., endothelial or T-cell specific) P55 deletions and combinations of different cre lines. On the basis of the remarkable reduction of TNF-induced inflammation observed when P55 levels are downregulated, we believe that P55 ASOs could be useful not only in combination of TNF/IFNg but also on their own as a treatment for TNF-mediated diseases such as arthritis and IBD. The TNF signaling pathway is a well-known mediator in several autoimmune diseases, and treatment with ASOs targeting TNF
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18. Vandenabeele P, Declercq W, Beyaert R, et al. Two tumour necrosis factor receptors: structure and function. Trends Cell Biol 1995;5:392–9. 19. Rothe J, Lesslauer W, Lotscher H, et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 1993;364:798–802. 20. Stoelcker B, Ruhland B, Hehlgans T, et al. Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature. Am J Pathol 2000;156:1171–6. 21. Cummins LL, Owens SR, Risen LM, et al. Characterization of fully 20 -modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res 1995;23: 2019–24. 22. Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol 2010;50:259–93. 23. van Deutekom JC, Janson AA, Ginjaar IB, et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med 2007;357:2677–86. 24. Miller TM, Pestronk A, David W, et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study. Lancet Neurol 2013;12: 435–42. 25. Raal FJ, Santos RD, Blom DJ, et al. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebocontrolled trial. Lancet 2010;375:998–1006. 26. Gleave ME, Monia BP. Antisense therapy for cancer. Nat Rev Cancer 2005;5:468–79. 27. Granger DL, Hibbs JB, Jr, Broadnax LM. Urinary nitrate excretion in relation to murine macrophage activation. Influence of dietary L-arginine and oral NG-monomethyl-L-arginine. J Immunol 1991;146:1294–302. 28. Van Snick J, Cayphas S, Vink A, et al. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor
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activity for B-cell hybridomas. Proc Natl Acad Sci USA 1986;83:9679–83. Huang XW, Yang J, Dragovic AF, et al. Antisense oligonucleotide inhibition of tumor necrosis factor receptor 1 protects the liver from radiation-induced apoptosis. Clin Cancer Res 2006;12:2849–55. Cauwels A, Van Molle W, Janssen B, et al. Protection against TNF-induced lethal shock by soluble guanylate cyclase inhibition requires functional inducible nitric oxide synthase. Immunity 2000;13:223–31. Smith JW, II, Urba WJ, Clark JW, et al. Phase I evaluation of recombinant tumor necrosis factor given in combination with recombinant interferon-gamma. J Immunother 1991;10:355–62. Teplova M, Minasov G, Tereshko V, et al. Crystal structure and improved antisense properties of 20 -O-(2-methoxyethyl)-RNA. Nat Struct Biol 1999;6:535–9. Walder RY, Walder JA. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA 1988;85:5011– 15. Geary RS, Watanabe TA, Truong L, et al. Pharmacokinetic properties of 20 -O-(2-methoxyethyl)modified oligonucleotide analogs in rats. J Pharmacol Exp Ther 2001;296:890–7. Agrawal S, Temsamani J. Comparative pharmacokinetics of antisense oligonucleotides. Methods Mol Med 1996;1:247–70. Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009;8:129–38. Myers KJ, Murthy S, Flanigan A, et al. Antisense oligonucleotide blockade of tumor necrosis factor-alpha in two murine models of colitis. J Pharmacol Exp Ther 2003;304:411–24. Bongartz T, Sutton AJ, Sweeting MJ, et al. AntiTNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. JAMA 2006;295:2275–85. Van Hauwermeiren F, Vandenbroucke RE, Libert C. Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1. Cytokine Growth Factor Rev 2011;22:311–19.
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and progression. Nat Rev Immunol 2005;5:749– 59. Van Hauwermeiren F, Armaka M, Karagianni N, et al. Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium. J Clin Invest 2013;123:2590–603. Senzer N, Mani S, Rosemurgy A, et al. TNFerade biologic, an adenovector with a radiationinducible promoter, carrying the human tumor necrosis factor alpha gene: a phase I study in patients with solid tumors. J Clin Oncol 2004;22: 592–601. Thamm DH, Kurzman ID, Clark MA, et al. Preclinical investigation of PEGylated tumor necrosis factor alpha in dogs with spontaneous tumors: phase I evaluation. Clin Cancer Res 2010;16: 1498–508. Brouckaert P, Ameloot P, Cauwels A, et al. Receptor-selective mutants of tumour necrosis factor in the therapy of cancer: preclinical studies. Circ Shock 1994;43:185–90. Gerspach J, Pfizenmaier K, Wajant H. Improving TNF as a cancer therapeutic: tailor-made TNF fusion proteins with conserved antitumor activity and reduced systemic side effects. Biofactors 2009; 35:364–72. Curnis F, Sacchi A, Borgna L, et al. Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat Biotechnol 2000; 18:1185–90. Wielockx B, Lannoy K, Shapiro SD, et al. Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy. Nat Med 2001;7:1202–8. Takahashi N, Vanlaere I, de Rycke R, et al. IL-17 produced by Paneth cells drives TNF-induced shock. J Exp Med 2008;205:1755–61. Duprez L, Takahashi N, Van Hauwermeiren F, et al. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity 2011;35:908–18. Huys L, Van Hauwermeiren F, Dejager L, et al. Type I interferon drives tumor necrosis factor-induced lethal shock. J Exp Med 2009;206: 1873–82.
ASOs prevent toxicity of TNF/IFNg treatment
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Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNc treatment in mouse tumor models Filip Van Hauwermeiren1,2, Roosmarijn E. Vandenbroucke1,2, Lynda Grine1,2, Leen Puime`ge1,2, Elien Van Wonterghem1,2, Hong Zhang3 and Claude Libert1,2 1
Inflammation Research Center (IRC), VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 3 Isis Pharmaceuticals, Carlsbad, CA 2
Cancer Therapy
Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55-deficient mice. In previous work we demonstrated that P551/2 mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNc) remain fully functional in these mice. Hence, a high dose of TNF/IFNc has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNc treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNc antitumor therapy.
Tumor necrosis factor (TNF) is a pleiotropic cytokine with an important role in diverse cellular processes, such as proliferation, differentiation, cell death and cell survival. It is best known for its key role in many inflammatory and autoimmune diseases, such as Crohn’s disease and rheumatoid arthritis.1 However, TNF was originally identified and named after its potent antitumor effects, especially in combination with interferon gamma (IFNg) and/or chemotherapeutics.2 Unfortunately, owing to its strong proinflammatory actions, its injection also causes systemic inflammation that can lead to life-threatening shock.3 Because the anticancer therapeutic Key words: TNF/IFNg therapy, TNFR1, antisense oligonucleotides, toxicity Additional Supporting Information may be found in the online version of this article Conflict of interest: H.Z. was an employee of Isis Pharmaceuticals Grant sponsors: Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), Vlaamse Liga tegen kanker (VLK), Stichting tegen de Kanker, BOF-UGent, the Research Foundation Flanders (FWO Vlaanderen), the Interuniversity Attraction Poles Program of the Belgian Science Policy DOI: 10.1002/ijc.28704 History: Received 11 Oct 2013; Accepted 11 Dec 2013; Online 31 Dec 2013 Correspondence to: Prof. Claude Libert, VIB IRC UGENT, Technologiepark 927, 9000 Ghent, Belgium, Fax: 1003292217673, E-mail: [email protected]
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dose of TNF is close to the maximally tolerated dose (MTD), its current use in anticancer therapy is limited to local settings in which systemic TNF actions are prevented, such as in isolated limb perfusion. This locoregional administration of TNF is highly effective, induces limited toxicity and is used as limb-sparing treatment for in transit melanoma and soft tissue sarcoma.4,5 The antitumor effect of TNF/IFNg therapy is mediated by inhibition of avb3 integrin, which is specifically expressed by neovascular endothelium cells and allows them to attach to the extracellular matrix.6 Loss of adhesion of these endothelial cells causes cell death, increased vascular permeability and eventually complete destruction of the tumor neovasculature. Interestingly, TNF-induced inflammation and toxicity is caused by a signaling cascade that culminates in activation of MAPKs and transcription factors such as NFjB,7 leading to the induction of multiple toxic mediators and ultimately to TNF-induced lethal inflammation.8 Therefore, separating those two biological activities seemed possible and several strategies have been proposed by different research groups to limit the toxicity of TNF and to increase its therapeutic potential in antitumor treatment. Multiple TNF mutants or TNF fusion proteins have been produced to limit toxicity or increase TNF accumulation in tumor tissues, e.g., TNFerade (a radiation-inducible TNF gene),9 chemically modified TNF, TNF mutants with lower toxicity10–12 and TNF fusion proteins (e.g., NGR-TNF) to target TNF to the tumor neovasculature.13
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What’s new? Tumor necrosis factor (TNF) can produce potent antitumor effects as well as potentially life-threatening systemic inflammation, which has limited its therapeutic use. Different strategies to overcome systemic toxicity have been explored, including the inhibition of mediators of TNF-induced toxicity. Here, antisense oligonucleotides targeted against TNFR1 (P55), the primary mediating receptor in models of both acute TNF toxicity and TNF/IFNc antitumor activity, were found to effectively lower TNFR1 protein levels in select tissues and thereby reduce toxicity associated with TNF/IFNc therapy, without affecting antitumor activity. The findings provide new evidence for the applicability and safety of TNF/IFNc therapy.
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targeting SOD1,24 elevated plasma LDL levels by targeting apoB-10025 and cancer by targeting multiple oncogenes.26 In our study, treatment with P55-targeting ASOs resulted in a strong downregulation of P55 protein levels in liver, blood mononuclear cells and small intestine. This effect was sufficient to protect mice against TNF toxicity in both acute and antitumor settings in several tumor models.
Material and Methods Mice
Female C57BL6, NMRI Nu/Nu and B6CBAF1 mice 8–12 weeks of age (Janvier, France) were maintained in conventional, temperature-controlled, air-conditioned animal houses with 14–10 hr light/dark cycle, and received food and water ad libitum. All experiments were approved by the local ethics committee. Oligonucleotide treatments
Mice were injected i.p. every other day for a total of four times with 25 mg/kg 20 -O-(2-methoxy)ethyl-modified ASO specific for P55 (ISIS 108426) or nonspecific control (ISIS 141923) dissolved in 200 ml phosphate-buffered saline (PBS). For the ASO safety studies in an antitumor setting, ASOs were injected i.p. at 25 mg/kg on days –7, –5, –3, –1, 1, 3, 5, 7 and 9 relative to the start of TNF/IFNg treatment. The ASOs were a kind gift from Isis Pharmaceuticals. P55 ELISA
Whole organ samples were isolated for kidney, spleen, liver and lung. We isolated intestinal epithelial cells by scraping them from different parts of the small intestine. Mononuclear cells were isolated after separation of 300 ml blood on a Ficoll gradient. Samples were homogenized in ice-cold buffer [PBS, 0.5% CHAPS and protease inhibitors (Complete, Roche, Vilvoorde, Belgium)]. Homogenates were centrifuged for 30 min at 20,000g and 4 C and the supernatant was stored at –80 C. Protein concentration was determined by the Bradford method (BioRad, Eke, Belgium). P55 levels were determined with the Quantikine sP55 ELISA kit (R&D Abingdon, UK). The levels were normalized to the P551/1 levels of untreated samples, which were set as 100%. Cytokines
Recombinant mouse TNF and IFNg were expressed in Escherichia coli and purified in our laboratory. TNF had a specific
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Another possibility is to focus on the identification and inhibition of TNF-induced toxic mediators while maintaining TNF’s antitumor potential. Previously, we identified multiple TNF-induced toxic mediators, including several members of the family of matrix metalloproteinases,14 IL17,15 necroptosis16 and type I interferons.17 More recently, we identified P55 (TNFR1 and CD120a) as a promising target for overcoming the toxicity of systemic TNF antitumor treatment.8 TNF acts by binding two different receptors, P55 and P75 (TNFR2 and CD120b).18 P55 has important physiological functions, possesses anticancer activity and contributes to pathological inflammatory processes,19 whereas P75 has been implicated mainly in immune modulation.1 P55 is the main receptor involved in both the acute TNF toxicity model and the TNF/IFNg antitumor model. TNF-induced tumor regression and toxicity are absent in P55-deficient mice, whereas P75-deficient mice behave like wild-type mice in these models.20 In previous work we found that P551/2 mice have solid protection against TNF toxicity. In TNF/IFNg antitumor treatment, P551/2 mice resist TNF toxicity of doses up to tenfold the LD50 for P551/1 mice, but the antitumor effects remain intact. In response to TNF, P551/2 mice have a strongly reduced induction of inflammatory signaling pathways and much reduced production of several toxic mediators.8 Moreover, although we reported before that the liver is an essential target of TNF’s lethal actions,14 we recently identified P55 expression levels on intestinal epithelial cells (IECs) as crucial determinants of systemic TNF toxicity. P551/1 expression specifically in the IEC is sufficient to induce TNF toxicity, while P551/2 or P552/2 expression in IEC is protective against TNF toxicity.8 Here, we wanted to mimic the phenotype of P551/2 mice by downregulating P55 expression levels using antisense oligonucleotides (ASOs, Isis Pharmaceuticals, Carlsbad, CA). These ASOs are 20 -O-(2-methoxyethyl) chemically stabilized oligonucleotides which are nuclease resistant and show increased affinity for their target sequence compared to unmodified oligonucleotides.21 They bind to their target mRNA and modify its function by different mechanisms, e.g., inducing its degradation, causing exon skipping or blocking translation.22 ASO-based therapies are now in clinical trials for various pathologies and show promising results also for the treatment of Duchenne muscular dystrophy (DMD) by targeting dystrophin,23 amyotrophic lateral sclerosis (ALS) by
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activity of 1.0 3 109 IU/mg. IFNg had a specific activity of 1.1 3 109 IU/mg. Endotoxin in the cytokine preparations was below the detection limit of a Limulus amoebocyte lysate assay. Cytokines and LPS were diluted in endotoxin-free PBS (GIBCO, Invitrogen, Merelbeke, Belgium) immediately before injection.
percent tumor area was determined by photographing the surface of each lung lobe’s front and back. As these tumors were black, it was possible to measure their areas with ImageJ software. Percent tumor area was calculated as the number of “black tumor” pixels divided by the total number of pixels in the lung area.
Acute TNF-bolus model
Statistical analysis
Mice were injected i.p. with mouse TNF diluted in 200 ml of PBS. Lethality was monitored for 72 hr, after which no further deaths occurred and body temperatures of surviving mice returned to normal.
Survival curves (Kaplan–Meier plots) were compared using a log-rank test. Means 6 SD were compared with a Student’s t-test. Final lethality data were compared using a Fisher’s exact test. *, ** and *** represent 0.01 < p < 0.05, 0.001 < p < 0.01 and p < 0.001, respectively.
Temperature measurement and blood collection
Rectal body temperature was measured with a digital thermometer. Blood was taken with a glass capillary from the tail vein and allowed to clot for 30 min at 37 C. The clot was removed and serum was isolated by centrifugation at 20,000g for 10 min and stored at 220 C. NOx determination in serum samples
NOx concentration in the serum (sum of the stable NO metabolites nitrate and nitrite) was determined as described.27 Determination of IL-6 in serum
IL-6 was measured as described before, i.e., by using an IL-6dependent 7TD1 hybridoma cell line.28
Cancer Therapy
Tumor cell cultures and antitumor experiments Subcutaneous B16BL6 melanoma. The murine B16BL6
melanoma cell line was a gift from M. Mareel (Ghent, Belgium) by courtesy of I. Fidler (Dallas, USA). Cultured B16BL6 cells were washed thrice in PBS and counted. Their density was adjusted with PBS and 100 ll containing 6 3 105 cells was injected subcutaneously (s.c.) in the shaved right thigh of mice. Treatment was started 12 days later by s.c. injection with 0.1 ml of a mixture of TNF and IFNg next to the tumor. This treatment was repeated daily for 10 days. Lethality and tumor size index (TSI) were scored. TSI was defined as the product of longest diameter of the tumor and the diameter perpendicular to it (mm2). Subcutaneous Lewis lung carcinoma. LLC cells were washed
thrice with PBS and counted. Their density was adjusted with PBS and 100 ml containing 6 3 106 cells was injected s.c. in the shaved right thigh of mice. Treatment was the same as for the B16BL6 melanoma model. Lung colony model. B16BL6 melanoma cells were washed
thrice in PBS and counted. Their density was adjusted with PBS and 200 ml containing 5 3 104 cells was injected in the tail vein of the mice. Treatment was started 15 days later by i.p. injection of 0.3 ml of PBS containing 10 or 50 mg TNF. This treatment was repeated daily for 10 days. Lethality was scored during this period and at the end of the treatment the
Results ASO treatment reduces P55 protein levels in liver and small intestine
We first investigated which organs are affected by the P55 ASO treatment. We based our protocol on a published injection schedule that resulted in 50% downregulation of P55 mRNA expression in the liver.29 Mice were injected intraperitoneally (i.p.) with control (CTR) ASO or P55 ASO treatment (25 mg/kg) every other day, four times in total (Fig. 1a). Twenty-four hours after the fourth ASO treatment, mice were sacrificed, tissues were isolated and processed and P55 protein levels were measured by ELISA. Treatment with P55 ASO caused a reduction in P55 protein levels in the liver (50%), blood mononuclear cells (75%) and different parts of the small intestine (75%), but not in lung, spleen and kidney (Fig. 1b). ASO-mediated P55 downregulation protects against acute TNF toxicity
We studied the effect of P55 downregulation in the acute TNF bolus model. Mice were treated four times with control ASO or P55 ASO (25 mg/kg) every other day, and 24 hr after the fourth treatment they were injected i.p. with 25 mg TNF, the LD100 for untreated animals (Fig. 2a). TNF injection is known to cause acute and lethal inflammatory shock associated with hypothermia, hypotension and abundant release of cytokines.8 Mice pretreated with P55 ASOs experienced milder hypothermia and were significantly protected against death (30% lethality) compared to CTR ASO-treated mice (80% lethality) (Figs. 2b and 2c). In a separate experiment, blood was collected from mice before and 3 hr after TNF injection, serum was prepared and TNF-induced NO metabolites and IL6 levels were measured. Mice treated with P55 ASO had significantly lower levels of NO metabolites (Fig. 2d) and serum IL6 (Fig. 2e) compared with CTR ASOtreated mice. P55 ASO treatment protects against TNF/IFNc toxicity in the B16Bl6 melanoma tumor model
In contrast to the acute TNF model, which is based on injection of a single bolus, our established and validated TNF/ C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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IFNg antitumor model requires daily TNF/IFNg injections for 10 days to induce complete tumor regression.2,8 We first used the B16BL6 melanoma model in which tumor cells are injected in the right thigh of mice and left to grow for 10 days to a size of about 1 cm2. Mice were injected i.p. every other day nine times with control ASO or P55 ASO (25 mg/ kg), four times during tumor growth and five times during TNF/IFNg treatment (Fig. 3a). No significant effects on tumor growth were observed when comparing P55 ASO with CTR ASO or PBS treatment (data not shown). Before the start of the actual TNF/IFNg treatment, P55 protein levels were measured in the liver after each ASO injection to confirm that the effect of ASOs on P55 was not influenced by growing tumors. Similar to the four times P55 ASO treatment described above, a stable downregulation of about 50% was observed in the livers of mice treated with P55 ASO throughout the whole period of tumor growth. In contrast, CTR ASO treatment had no effect on P55 levels. P55 levels in the inoculated tumors were also measured during tumor growth, but they were not affected by P55 ASO treatment, which is appropriate for a full P55-mediated antitumor response (Figs. 3b and 3c). In the next experiment, mice pretreated with ASOs were injected daily with PBS or 17.5 mg TNF and 5,000 IU IFNg C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
for 10 days and tumor size and lethality were monitored daily. After 10 days of TNF/IFNg treatment, death of mice was monitored for 10 days and then the survivors were euthanized. CTR ASO or P55 ASO treatment on its own did not influence tumor growth in the PBS-treated groups. Daily TNF/IFNg treatment caused strong tumor regression in mice cotreated with CTR ASOs or P55 ASOs (Fig. 3d). However, P55 ASO cotreated mice were significantly protected against the toxic effects and survived the antitumor treatment, in contrast to CTR ASO cotreated animals, all of which died from TNF toxicity during the study (Fig. 3e). Similar results using the B16Bl6 model were obtained in genetic backgrounds other than C57BL/6, namely NMRI Nu/Nu and B6CBAF1 mice (Supporting Information Figs. 1A–1D). P55 ASO treatment protects against TNF/IFNc toxicity in other tumor models
To confirm the therapeutic value of P55 ASO cotreatment, two other cancer models were used. In both models TNF/ IFNg and ASO cotreatment was identical to the treatment schedule used for the B16Bl6 tumor model (Fig. 3a). One model was the Lewis lung carcinoma (LLC) tumor model.30 LLC cells were injected in the right thigh of mice and the tumors were allowed to grow to about 1 cm2. Tumor-bearing
Cancer Therapy
Figure 1. Effects of P55 ASO treatment on P55 protein levels in multiple organs. (a) ASO pretreatment schedule. Mice were pretreated four times with 25 mg/kg CTR or P55 ASO. Twenty-four hours after the last pretreatment mice were sacrificed and organs were collected. (b) P55 ELISA after treatment with P55ASO. Mice were treated with P55 ASO (n 5 5) or control ASO (n 5 5). P55 ASO resulted in significant downregulation of P55 in the liver, small intestine and blood mononuclear cells compared to CTR ASO, but no difference was observed in P55 levels in lung, spleen and kidney.
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Cancer Therapy
Figure 2. P55 ASO treatment protects against acute TNF toxicity. (a) ASO pretreatment schedule. Mice were pretreated four times with 25 mg/kg CTR or P55 ASO. Twenty-four hours after the last pretreatment mice were injected i.p. with 30 mg TNF. (b and c) Survival after acute TNF treatment. Mice treated i.p. with P55 ASO (green, •, n 5 19) were significantly protected against a single injection of TNF compared to control ASO-treated mice (red, 䊏, n 5 15) and experienced milder hypothermia (c). (d and e) NOx levels and IL6 levels 3 hr after i.p. injection of 30 mg TNF in P55 ASO-treated mice. Induction of IL6 and NOx was weaker in P55 ASO-treated mice (n 5 10) than in mice treated with control ASO (n 5 9).
mice were then pretreated with CTR or P55 ASOs as described for the B16Bl6 model, and the tumors were injected paralesionally with 17.5 mg TNF and 5,000 IU IFNg. Again, P55 ASO cotreatment allowed full antitumor effects and resulted in significantly reduced toxicity (only 10% lethality in contrast to 90% in CTR ASO-treated mice) (Figs. 4a and 4b). We also used the B16Bl6 lung colony model. Intravenous injection of 5 3 104 melanoma cells resulted in the formation of lung nodules, which were allowed to grow for 15 days. Then, mice were treated i.p. with 15 mg TNF and 5,000 IU IFNg daily for 10 days. Only mice surviving the 10-day treatment were considered for tumor growth evaluation. Tumor burden was analyzed by calculating the percentage of black melanoma surface area on the individual lung lobes. Compared to the PBS-treated groups, the TNF/IFNg-treated groups showed reduced tumor burden: 10% reduction for the CTR ASO cotreated mice and 18% reduction for the P55 ASO cotreated mice. P55 ASO cotreated mice were protected
against TNF/IFNg treatment and all of them survived the treatment, whereas only 45% of CTR ASO cotreated mice survived (Figs. 4c and 4d). Calculation of the maximum tolerable dose in P55 ASO-treated mice
Finally, we determined the toxicity profile of daily TNF/IFNg therapy for 10 days in B16Bl6 tumor-bearing mice cotreated with control ASOs or P55 ASOs according to the same injection schedule (Fig. 3a). Tumors were inoculated and left to grow for 10 days, after which mice were injected daily with TNF (doses ranging from 10 to 25 mg per day per mouse), combined with 5,000 IU IFNg. Death of mice was followed during the 10 days of therapy with TNF/IFNg and during a follow-up period of 10 days. After that the remaining mice were euthanized. Treatment with CTR ASO resulted in an LD50 of 15 mg TNF, while P55 ASO-treated mice reached a 50% higher LD50 of 22.5 mg TNF (Figs. 5a and 5b and Supporting Information Fig. 2). C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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Discussion TNF possesses potent antitumor properties but their full exploitation is hampered by its strong proinflammatory effects. Nevertheless, the clinical use of TNF, along with studies in mice, supports the view that TNF has spectacular anticancer effects on a wide variety of solid tumors.2,5,31 We recently identified P55 as an excellent therapeutic target to reduce the toxicity of systemic TNF/IFNg antitumor therapy.8 P551/2 mice are solidly protected against TNF toxicity in TNF/IFNg antitumor models, while retaining the potent TNF-induced antitumor effects. To provide proof of principle for pharmacologic treatment, we used P55 targeting ASOs to mimic the P551/2 situation. We made use of the second-generation ASOs, 20 -Omethoxyethyl (20 MOE)-modified ASOs (Isis Pharmaceuticals), which have increased stability owing to their RNase resistance C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
and increased affinity for target mRNA compared to the firstgeneration phosphorothioate oligonucleotides.32 ASOs can strongly reduce protein levels of a target gene, e.g., by recognition of the mRNA–ASO complex, resembling a DNA–RNA intermediate, which gets selectively degraded by RNase H.33 However, the effectiveness of therapy largely depends on delivery to the desired target organ or cell type. Pharmacokinetic studies indicate that systemic delivery of naked ASOs leads mainly to liver and kidney targeting whereas spleen and intestine are targeted with lower efficacy.34,35 In our study, we observed that systemic treatment with P55 ASO reduces P55 protein levels in liver, blood mononuclear cells and IECs but not in lung, kidney, spleen or tumor tissue. Interestingly, treatment with P55-specific ASOs protected against acute TNF toxicity. Upon TNF injection, mice treated with P55 ASO had significantly better survival, a
Cancer Therapy
Figure 3. P55 ASO treatment protects against TNF toxicity in TNF/IFNg antitumor treatment. (a) ASO pretreatment and TNF/IFNg injection schedule. Mice were inoculated with 6 3 105 B16Bl6 cells on day –10 and treated nine times with 25 mg/kg CTR or P55 ASO starting on day –9. Daily treatment with TNF/IFNg started on day 0. (b and c) P55 ELISA after treatment of tumor-bearing mice with ASO but without TNF/IFNg treatment. Liver P55 levels in P55 ASO-treated mice (n 5 4 per time point) were significantly reduced compared to control ASOtreated mice. No significant downregulation of P55 was found in tumor tissue after treatment with P55 ASO. (d and e) Antitumor effects and survival of ASO-treated mice in an antitumor experiment. The P55 ASO-treated mice (•, n 5 10) had better survival than control ASO mice (䊏, n 5 10) when treated daily with 17.5 mg TNF and 5,000 IU IFNg. No significant differences between the groups were found in tumor regression and tumor growth (empty symbols stand for PBS and filled symbols stand for TNF/IFNg treatment).
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Figure 4. P55 ASO treatment protects against TNF/IFNg toxicity in LLC and B16Bl6 lung colony model. (a and b) Antitumor effects (a) and survival (b) of ASO-treated mice in a LLC antitumor experiment. Mice were first inoculated s.c. with 6 3 106 LLC cells and then treated with ASOs as described for the B16BL6 model above. Finally, they were treated for 10 days with 17.5 mg TNF 1 5,000 IU IFNg or with PBS. Tumors regressed well in all groups but only the P55 ASO-treated mice (green, • n 5 9) had significantly better survival than the control ASO-treated mice (red, 䊏, n 5 9). (c and d) Antitumor effects and survival of ASO-treated mice in the B16Bl6 lung colony model. Mice were injected i.v. with 50,000 B16Bl6 melanoma cells, and after 15 days TNF/IFNg or PBS treatment was initiated. There was a small reduction of tumor area in all TNF/IFNg-treated groups but P55 ASO (n 5 8)-treated mice had the better survival compared to CTR (n 5 9).
milder drop in body temperature and weaker induction of the inflammatory mediators IL6 and NO. This protection was also observed in the TNF/IFNg antitumor model: P55 ASO-treated mice had the best outcome, with remarkable tumor regression and significantly better survival than the PBS- or CTR ASO-treated mice. Such tumor regression without the associated TNF toxicity was also obtained in different tumor models as well as in different genetic backgrounds, indicating that it might be a widely applicable therapeutic option. Finally, P55 ASO therapy increased the maximum tolerated TNF dose (MTD) by 50% compared to CTR ASOtreated mice. These data provide proof of principle that P55
ASO treatment reduces P55 expression and increases the therapeutic potential of TNF in cancer therapy. Although MTD increased 50% by P55 ASO therapy, we could not mimic the P551/2 phenotype completely, which had a tenfold higher MTD.8 This is likely due to nonsystemic P55 downregulation in a rather limited set of organs (liver, IECs and blood mononuclear cells). Better results can be expected when P55 protein levels are also downregulated in other cell types that mediate TNF toxicity. To increase the uptake in target tissues different strategies can be used. Modifications can be used to optimize the delivery of ASOs to the target tissues: liposome encapsulation, cholesterol modification C 2013 UICC Int. J. Cancer: 135, 742–750 (2014) V
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Figure 5. P55 ASO pretreatment increases the maximum tolerable dose of TNF in a TNF/IFNg antitumor treatment. (a) Lethality data from the toxicity study of TNF/IFNg-treated B16BL6-inoculated mice treated with CTR ASOs or P55 ASOs. (b) Summary of the lethality data. LD50 was 15 mg in CTR ASO-treated mice and 22.5 mg in P55 ASO-treated mice.
can protect mice in a model of colitis.37 P55 inhibition might be an interesting alternative to current anti-TNF therapies. By specifically inhibiting P55 signaling instead of completely blocking TNF, the often beneficial and immune modulatory P75 signaling is kept intact, and this might enhance repair functions and reduce anti-TNF-associated side effects, as observed, e.g., in multiple sclerosis.1,38,39 In summary, we demonstrate that reduction of P55 expression by P55 ASO increases the safety of TNF in anticancer therapy without loss of anticancer efficacy. There is still room for improvement by using better delivery systems for broader downregulation of P55 protein expression in other tissues that mediate TNF toxicity. Identification of these tissues and development of strategies to target them will further increase the MTD of TNF and might allow broader applicability and systemic exploitation of the remarkable antitumor effects of TNF therapy.
Acknowledgements This work was supported by grants to CL from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWTVlaanderen), Vlaamse Liga tegen kanker (VLK), Stichting tegen de Kanker, BOF-UGent, the Research Foundation Flanders (FWO Vlaanderen) and the Interuniversity Attraction Poles Program of the Belgian Science Policy. The authors wish to thank Dr. Amin Bredan for editing the manuscript. They also thank Isis Pharmaceuticals for providing antisense oligos and Wilma Burm and Joke Vanden Berghe for excellent technical assistance. CL and FVH formulated the hypothesis and initiated and organized the study. FVH, REV, LP, LG and EVW performed the experimental work and analyzed the data. CL oversaw the experiments and data. HZ and CL provided essential materials and funding. FVH drafted the manuscript and CL finalized it.
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ASOs prevent toxicity of TNF/IFNg treatment
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