Piotr Jachimczak

Transforming growth factor-beta-mediated autocrine growth regulation of gliomas as detected with phosphorothioate antisense oligonucleotides

Transforming growth factors‐β1 and ‐β2 (TGF‐β1 and ‐β2) are important growth‐regulatory proteins for astroglial neoplasms. We analyzed their role in tumor‐cell proliferation in 12 glioma cell lines, employing phosphorothioate antisense oligodeoxynucleotides (S‐ODNs, 14 mer), specifically targeted against the coding sequences of TGF‐β1‐mRNA and TGF‐β2‐mRNA. TGF‐β1‐S‐ODNs inhibited cell proliferation in 5 of 12 gliomas, whereas TGF‐β2‐S‐ODNs reduced the cell proliferation in all glioma cell lines, compared to nonsense‐S‐ODN‐treated and S‐ODN‐untreated cells as controls. The efficacy and specificity of antisense effects was validated by Northern‐blot analysis and determination of protein concentrations in culture supernatants (ELISA). Exogenous hrTGF‐β1 either stimulated or inhibited the cell lines, whereas pnTGF‐β2 stimulated the proliferation of most glioma cells. Blocking the extracellular pathway of TGF‐β by neutralizing antibodies only slightly inhibited those cell lines, which were markedly stimulated by TGF‐βs. As the effects of TGF‐β2‐S‐ODNs were much stronger than those of TGF‐β neutralizing antibodies, we postulate that the endogenously produced TGF‐β2 control glioma‐cell proliferation, in part by an intracellular loop.

The Antisense Oligonucleotide Trabedersen (AP 12009) for the Targeted Inhibition of TGF-β2

Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-β2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors. Here we describe the development of the antisense phosphorothioate oligodeoxynucleotide trabedersen (AP 12009) which was designed for the specific inhibition of TGF-β2 biosynthesis. In vitro and in vivo experiments confirmed the mode of action, efficacy and tolerability of trabedersen and paved the way for clinical studies. In patients with high-grade glioma, intratumoral treatment with trabedersen is currently evaluated in a pivotal, randomized and active-controlled phase III study. Intravenous application of trabedersen for the treatment of patients with advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma is assessed in a currently ongoing phase I/II dose escalation study.

Treatment of malignant gliomas with TGF-beta2 antisense oligonucleotides

Antisense oligodeoxynucleotides (AS-ODNs) have been widely used to determine gene function, validate drug targets and as novel therapeutics for human diseases. In this review, we describe the development of AS-ODNs, including their modifications, pharmacokinetics and toxicity in animal models and humans, and their preclinical and clinical development in the therapy of human high-grade gliomas. The most advanced AS-ODN for the therapy of high-grade gliomas is a phosphorothioate-modified AS-ODN, AP 12009 (trabedersen), which targets mRNA encoding TGF-β2. AP 12009 is administered intratumorally using convection-enhanced delivery. A series of Phase I and II clinical trials have evaluated the toxicity profile and optimal dose of the substance. A randomized, controlled international Phase III study was initiated in March 2009 and will compare trabedersen 10 µM versus conventional alkylating chemotherapy in patients with recurrent or refractory anaplastic astrocytoma after standard radio- and chemotherapy.

Inhibition of immunosuppressive effects of melanoma-inhibiting activity (MIA) by antisense techniques

Melanoma inhibitory activity (MIA) is an 11 kD protein secreted by malignant melanomas. Recent studies revealed an interaction of MIA with epitopes of extracellular matrix proteins including fibronectin. Structural homology of MIA with the binding sites of α4β1 integrin results in complex interactions of MIA with molecules binding to α4β1 integrin. As cells of the immune system express α4β1 integrins (VLA‐4), we investigated whether MIA may modulate the function of human leukocytes. Here we describe the effects of MIA on the activation of human PBMCs and auto‐/allogeneic lymphokine‐activated killer cell (LAK) cytotoxicity in human MIA‐negative glioma cell lines and MIA‐positive melanoma cell lines in vitro. MIA inhibits PHA‐ or IL‐2‐induced human PBMC proliferation in a dose‐dependent manner up to 63% (3H‐Tdr incorporation) and 59% (cell count), respectively, when added to the cell culture prior to mitogen stimulation. In addition, both autologous (GL and HW) and allogeneic (HTZ‐17, HTZ‐243 and HTZ‐374) antitumor LAK cytotoxicity was reduced by the addition of exogenous rhMIA (500 ng/ml, f.c.). Consequently, endogenous inhibition of MIA expression in human melanoma cells by MIA‐specific phosphorothioate antisense oligonucleotides enhanced the autologous LAK‐cell activity to the same level as observed in MIA‐negative human HMB melanoma cells expressing an MIA‐antisense construct. Our results indicate that MIA may contribute to immunosuppression frequently seen in malignant melanomas by inhibiting cellular antitumor immune reactions. Antagonization of MIA activity using antisense techniques may represent a novel therapeutic strategy for treatment of malignant melanomas.

Intrathecal treatment of C6 glioma leptomeningeal metastasis in Wistar rats with interlenkin-2

SummaryThe efficacy of intrathecal treatment of leptomeningeal metastasis (LM) with interleukin-2 (IL-2) was evaluated in an animal model using Wistar rats inoculated intracisternally with 107 C6 glioma cells. Prior to the in vivo experiments the antiproliferative effects of human IL-2, and of murine IFN-γ and TNF-α which are cytokines induced by IL-2 were tested in a colony forming assay. Only IFN-γ caused a dose-dependent inhibition of colony formation. Twelve animals were treated intracisternally with either 105 IU IL-2 or control medium on day 0, 2, and 5 after tumor cell inoculation. Both IL-2 treated and sham-treated animals developed LM with a symptom-free survival of 7 to 9 days. There was no significant difference between treated and untreated animals regarding time to onset of symptoms and pattern of tumor growth. Infiltration of the tumor tissue with ED-1+ monocytes and macrophages, and CD8+ lymphocytes, however, was slightly increased in IL-2 treated animals. In a second experiment 4 non tumor-bearing Wistar rats were intracisternally injected with a single dose of 105 IU IL-2. These animals also showed slightly enhanced leptomeningeal infiltration with CD8+ lymphocytes compared to controls. We conclude that intrathecal application of high-dose IL-2 although eliciting a slight immune reaction within the leptomeninges does not inhibit leptomeningeal tumor growth or prolong symptom-free survival in our animal model of LM. These results raise doubt about the clinical efficacy of intrathecal IL-2 treatment in patients with LM.

Contrast-Enhanced Transcranial Duplex Sonography

Introduction Transcranial Duplex Sonography (TDS, synonym: transcranial colour coded real time sonography, TCCS) has been further improved: aside from vascular and parenchymal anatomy, a broad spectrum of intracerebral pathology may be disclosed. Transpulmonary stable ultrasound contrast agents further improve image quality and signal intensity, with a broad spectrum of new applications. A synopsis of the applications of galactose-based contrast agents may be found in the review by Schlief et al [Schlief, 1993, #16]. Aside from a complete depiction of the arterial system and the basal veins, candidates for contrast-enhanced transcranial sonography may be mainly lesions with low blood flow velocities. There are advantages over conventional pulsed transcranial sonography (TCD): (1) rapid identification of large intracranial vessels within the B-mode image, resulting in shorter examination times and improved standardisation and reproducibility; (2) real time localisation of vascular pathology by colour imaging within the black and white B-mode image of the parenchyma, and (3) detection of parenchymal lesions with new diagnostic information, complementing MRIand CT-findings [Becker, 1992, #4].

The role of transforming growth factor-β in carcinogenesis

In their review, Gleave and Monia1 gave an overview of the current status and future directions of selected antisense compounds in clinical development for systemic cancer therapy. We would like to add some points to the discussion, as to the clinical potential of first-generation phosphorothioate oligodeoxynucleotides (PS-ODNs). Taking Gentas Genasense as an example, evidence of effectiveness, indicated by a marked increase in progression-free survival and responses in melanoma, was demonstrated2. The criticism of the Food and Drug Administration (FDA) during the Oncology Drugs Advisory Committee (ODAC) meeting was directed at study design deficiencies, not to the nature of the compound. Nevertheless, Genasense is still in advanced clinical trials and has recently shown activity for non-Hodgkin lymphoma3. Failure to meet the primary endpoint in melanoma could have several causes; the chemical nature of the compound is just one suggested reason. If the chemical modification was the reason for the lack of efficacy of antisense compounds in recent clinical trials as cited in the review, the logical consequence would be to target molecules such as protein kinase C α (PKCα) or BCL2 with second-generation ODNs. Instead, the compounds in current clinical development that were suggested as promising by the authors are directed against other targets. Consequently, the change in targets makes the comparison between the ODN-generations regarding anti-tumour efficacy impossible. In our opinion, the choice of target is most important for successful antisense therapy. The ideal drug candidate should drive tumour progression and should not, as is the case for PKCα, have redundant pathways. The crucial role in cancer of transforming growth factor β (TGFβ) has recently been the topic of numerous publications4–10 and conferences devoted to the TGFβ superfamily (American Association for Cancer Research, 2003, Keystone Symposium, 2005 and Federation of American Societies for Experimental Biology, 2005). So, we chose a target with pleiotropic effects — TGFβ overexpression in advanced tumours has been shown to correlate with tumour-induced immunosuppression, invasiveness and angiogenesis, and is associated with malignant progression. Blocking TGFβ therefore constitutes a multimodal antitumour approach. AP 12009 is a PS-ODN, referred to in the review as a first-generation PS-ODN, for the targeted downregulation of TGFβ2. Although the authors argue that the first-generation ODNs were the reason for current lack of clinical success, including insufficient potency, low stability and increased side effects compared to second-generation ODNs, we could show an excellent safety profile of this first-generation drug in both animal systemic toxicological studies11 and phase I/II studies in high-grade glioma patients with local application to circumvent the blood–brain barrier12–14 (P. Hau and K.J., personal communication). Furthermore, anti-tumour activity in phase I/II studies demonstrated TGFβ2 suppression with AP 12009 to be a promising therapeutic approach. A phase I/II study for the intravenous treatment of patients with other solid tumours is ongoing. The future therapeutic success of antisense compounds will depend, as is the case with any targeted therapy, on the careful selection of optimal targets, dosing, schedules and clinical trial design. A comparison of clinical efficacy of antisense compounds with different chemical modifications for the same target is neither given nor suggested for future studies in the review. So, the judgment on the therapeutic usefulness of different chemical modifications of antisense compounds as implied in the review cannot be made based on comparing the results of one chemical modification with a particular sequence against a particular target (for example ISIS 3521/Affinitak against PKCα ) with the results of another chemistry with a different sequence against a different target gene (for example OGX-011 against clusterin).

Abstract 301: RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination

Novel anti-cancer immune therapeutic strategies, like various new antibody formats and/or tumor vaccines, show promising results in patients. However, due to the “immune-escape phenomenon” driven by tumor-secreted Transforming Growth Factor-beta (TGF-beta), the host immune system of cancer bearing patients frequently fails to control tumor re-growth. The RealTVac® approach aims at avoiding this “immune-escape phenomenon” by intratumoral inhibition of active TGF-beta isotypes while simultanously and synergistically inducing an efficient immune response by a highly potent combination of immune stimulating factors. The synergistic effects of the proposed combination of a TGF-beta inhibitor with immunostimulating cytokines upon human immune cell activation are demonstrated in in-vitro experiments. Both, immune cell proliferation and tumor cell cytotoxicity were significantly enhanced. Initial experiments in a syngenic B16 melanoma xenograft model in immunocompetent mice indicate that the RealTVac® therapy of established subcutaneous B16 tumor xenografts reduced local tumor growth compared to untreated controls. To summarize, the goal of the local application of RealTVac® is to allow the host immune system to scan in real-time all Tumor Associated Antigens (TAAs) being currently expressed by those tumor cells that are exposed to sufficient TGF-beta inhibition. The repetitive administration of the RealTVac® approach is furthermore expected to provide a continuous update of the immune response, reflecting the changing pattern of TAAs related to the dynamic intratumoral heterogeneity in malignant tumors. Citation Format: Piotr Jachimczak, Andreas Mitsch, Achim Aigner. RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 301. doi:10.1158/1538-7445.AM2015-301

Abstract 3716: Targeted therapy of high-grade gliomas using TGF-beta2 inhibitor trabedersen (AP 12009): Results of the Phase IIb study as basis for the Phase III SAPPHIRE Study

Background: High-grade gliomas (HGG) strongly overexpress TGF-beta 2. Trabedersen, a phosphorothioate antisense oligonucleotide is a TGF-beta 2 inhibitor administrated intratumorally using convection-enhanced delivery (CED). So far, clinical studies in HGG have included three Phase I/II studies and one randomized, active-controlled, open-label, multinational dose-finding Phase IIb study. Methods: Main objectives of the Phase IIb study were to compare response rate, survival, and safety of 2 doses of trabedersen (10 µM or 80 µM) vs. standard chemotherapy (TMZ or PCV). 145 patients with recurrent or refractory HGG (AA WHO grade III or GBM WHO grade IV) were randomized. Trabedersen was given intratumorally by convection-enhanced delivery via a single multi-hole catheter with up to 11 treatment cycles (7-d-on, 7-d-off / cycle). Results: As in the previous Phase I/II studies, treatment with trabedersen led to long-lasting remissions in patients with recurrent or refractory AA or GBM. Trabedersen showed a good tolerability and safety profile. Overall, 10 µM was superior to 80 µM trabedersen in both efficacy and safety. Overall, the highest efficacy was observed in AA patients treated with the lower dose of trabedersen. The 10 µM trabedersen group showed a consistently higher survival rate compared to 80 µM trabedersen and standard chemotherapy (at 24 months: 83.3%, 53.3% and 41.7%, respectively). Duration of response was about 3 times longer in the 10 µM trabedersen group (29.1 months) compared to the standard chemotherapy group (8.0 months). The median overall survival was higher in both trabedersen groups than in the chemotherapy control group, with a remarkable survival benefit of 10 µM trabedersen over chemotherapy of 17.4 months. In GBM patients, trabedersen was as efficacious as standard chemotherapy. Furthermore, in a prespecified subgroup (age ≤55 years, KPS >80%), trabedersen patients had a markedly higher 2-year survival rate compared to standard chemotherapy (40.0 vs. 13.3%). Conclusion and Outlook: Trabedersen treatment is safe and has a clear clinical benefit in HGG. A randomized, multinational and active-controlled Phase III study (SAPPHIRE) with trabedersen as monotherapy in patients with recurrent or refractory AA (WHO grade III) has started and patient enrollment is ongoing. In this pivotal study, patients are randomized either to treatment with 10 µM trabedersen or to standard chemotherapy (TMZ or BCNU). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3716.

Treatment of Malignant Gliomas with Antisense Oligonucleotides

Antisense oligonucleotides (ODNs) are single-stranded, chemically-modified DNA-like molecules for the inhibition of gene translation by sequence-specific knockdown of mRNA through Watson–Crick hybridization. In general, their size ranges from 12 to 25 nucleotides in length, with the majority of ODNs being around 18–21 nucleotides. The natural phosphodiester backbone ODNs are rapidly degraded in biological fluids by ubiquitous nucleases. Therefore, a variety of heterocyclic modifications have been developed to strengthen base-pairing and thus stabilize the duplex formation between antisense ODN and their target mRNA. Antisense ODNs have been widely used for determining gene function, validation of drug targets and, finally, as novel therapeutics for human diseases. In this chapter, we will describe the development of antisense ODNs including their modifications, pharmacokinetics, and toxicity in animal models and humans, and their preclinical and clinical development in the therapy of human high-grade gliomas. The most advanced ODN in the therapy of high-grade gliomas is a phosphorothioate modified ODN (S-ODN), AP 12009 (trabedersen), which targets the mRNA encoding transforming growth factor beta2 (TGF-beta2). AP12009 is administered intratumorally using convection-enhanced delivery (CED). A randomized, controlled international Phase III study in recurrent or refractory anaplastic astrocytoma (SAPPHIRE) is planned to start in early 2009. Further antisense molecules targeting malignant glioma in clinical development are Affinitak, a PKC-alpha-S-ODN, and an ODN against IGF type I receptor for ex vivo use. In our opinion, antisense ODNs have potential for clinical applications in cancer patients even in a long-term perspective. They can be designed specifically against their target mRNA, are sufficiently stable in vivo and show first antitumor efficacy in human clinical trials with an excellent toxicity profile. Additionally, novel delivery techniques, like CED, may further improve their pharmacological profile making them superior to other DNA targeting strategies in humans.